Recombinant Lactobacillus johnsonii UvrABC system protein B (uvrB), partial

What is the UvrABC system and what role does UvrB play in Lactobacillus johnsonii?

The UvrABC system is a central nucleotide excision repair (NER) mechanism in prokaryotes, including Lactobacillus johnsonii. UvrB functions as the critical damage recognition component within this system, specifically recognizing and binding to DNA lesions. Within this repair system, UvrB works in coordination with UvrA and UvrC to excise bulky DNA lesions that can distort the DNA helix .

Methodologically, to study UvrB function in L. johnsonii:

• Isolate genomic DNA from L. johnsonii strains

• Amplify the uvrB gene using PCR with specific primers

• Clone the gene into an expression vector

• Express recombinant UvrB protein

• Conduct DNA binding assays to assess recognition functionality

How does the structure of UvrB relate to its function in DNA damage recognition?

UvrB contains several important structural features that facilitate DNA damage recognition:

• A β-hairpin motif that intrudes between DNA strands at damage sites

• A helicase domain that aids in DNA unwinding

• Domain 1B which forms a tunnel with the β-hairpin for DNA translocation

• A hydrophobic pocket behind the β-hairpin that can accommodate flipped-out damaged bases

Crystal structures have revealed that UvrB's β-hairpin creates a physical barrier that can detect distortions in the DNA double helix. The protein has been shown to flip out bases on the damaged strand, with the flipped-out base stabilizing in a hydrophobic region of the UvrB protein .

What are the optimal vector systems for expressing recombinant UvrB in Lactobacillus johnsonii?

For successful expression of recombinant UvrB in L. johnsonii, researchers should consider:

• Vector choice: Modified pLp vectors with strong constitutive promoters (e.g., SlpA promoter) show optimal expression in Lactobacillus species

• Selection markers: Use erythromycin resistance for selection in L. johnsonii

• Transformation method: Electroporation with glycine-enhanced cells yields higher transformation efficiency

• Expression tags: C-terminal tags are preferable as N-terminal tags may interfere with DNA binding functionality

When expressing fusion proteins (as demonstrated in the TTmim-PrtB fusion protein studies), codon optimization for L. johnsonii is essential, with a preference for A/T richness at the third codon position to ensure proper translation efficiency .

How can the UvrB gene be modified to enhance expression or functional analysis in L. johnsonii?

Several strategic modifications can enhance UvrB expression and analysis:

• Codon optimization: Adjust codons to match L. johnsonii preferences (34.91% GC content)

• Promoter selection: Use strong native promoters from L. johnsonii such as those identified in strain ZLJ010

• Signal sequence modification: Add a secretion signal if extracellular localization is desired

• Fusion tags: Consider adding epitope tags (His, FLAG) for purification and localization studies

• Site-directed mutagenesis: Introduce specific mutations in key residues (e.g., Tyr92, Tyr93, Phe249) to analyze structure-function relationships

For functional analysis, compare DNA repair efficiency between wild-type and modified UvrB variants using UV sensitivity assays or specific DNA damage repair assays.

What experimental approaches best assess the DNA damage recognition mechanism of L. johnsonii UvrB?

To comprehensively evaluate UvrB's damage recognition mechanism:

In vitro approaches:

• Electrophoretic mobility shift assays (EMSA): Use synthetic DNA substrates containing specific lesions

• Fluorescence studies: Utilize 2-aminopurine (2-AP) positioned on damaged DNA to monitor base-flipping by UvrB

• Single-molecule techniques: Apply FRET or optical tweezers to observe UvrB-DNA interactions in real-time

• Molecular dynamics simulations: Model the interaction between UvrB and damaged DNA to predict structural changes

In vivo approaches:

• DNA damage sensitivity assays: Compare survival of wild-type and uvrB-mutant strains after exposure to DNA-damaging agents

• ChIP-seq: Map genome-wide UvrB binding sites following DNA damage

• Reporter assays: Develop systems to quantify repair efficiency in living cells

The choice of DNA lesions is critical—use well-characterized damages such as benzo[a]pyrene adducts, which are recognized by UvrB as demonstrated in molecular dynamics studies .

How can researchers investigate the interaction between UvrB and other components of the UvrABC system in L. johnsonii?

To study the UvrA-UvrB-UvrC interaction network:

• Co-immunoprecipitation (Co-IP): Use tagged versions of UvrA, UvrB, and UvrC to pull down protein complexes

• Bacterial two-hybrid systems: Assess direct protein-protein interactions

• Surface plasmon resonance (SPR): Measure binding kinetics between purified components

• Crosslinking mass spectrometry: Identify specific interaction domains

• Fluorescence microscopy: Track co-localization of fluorescently tagged proteins during DNA damage response

Recent structural studies have revealed the UvrA-UvrB interaction interface regulated by ATP . When designing experiments, consider:

• The role of ATP hydrolysis in complex formation and dissociation

• The sequential nature of the UvrABC system (UvrA-UvrB handoff followed by UvrC recruitment)

• The conformational changes that occur during damage recognition and verification

How does L. johnsonii UvrB differ from UvrB in other bacterial species, and what are the functional implications?

Comparative analysis reveals several key differences:

These differences may impact:

• Specificity for different types of DNA damage

• Efficiency of the repair process in different environments

• Interaction with species-specific variants of UvrA and UvrC

• Response to environmental stresses unique to L. johnsonii's ecological niche

Methodology for comparative studies should include phylogenomic analysis based on single-copy genes, as performed for other L. johnsonii proteins .

How can recombinant L. johnsonii UvrB be used to develop improved DNA repair models?

Recombinant L. johnsonii UvrB can advance DNA repair models through:

• Bacterial models for human NER disorders: L. johnsonii UvrB can serve as a model for understanding eukaryotic NER mechanisms, which are evolutionarily related

• Synthetic biology applications: Engineer bacteria with modified UvrB to enhance DNA repair capabilities

• Structural templates: Use L. johnsonii UvrB crystal structures to refine molecular models of damage recognition

• Biotechnology applications: Develop UvrB-based biosensors for detecting environmental DNA-damaging agents

The methodological approach would involve:

• Expressing recombinant UvrB variants with specific mutations

• Testing their function in DNA binding and repair assays

• Correlating structural changes with functional outcomes

• Developing computational models that predict repair efficiency

What potential applications exist for engineering L. johnsonii strains with modified UvrB for research purposes?

Modified UvrB in L. johnsonii enables several research applications:

• Improved mucosal vaccine delivery systems: L. johnsonii with enhanced DNA repair capabilities could provide more stable vaccine delivery platforms

• Study of host-microbiome interactions: Modified strains can help understand how bacterial DNA repair affects persistence in the host

• Probiotic enhancement: Engineering UvrB to improve stress resistance could yield more robust probiotic strains

• Genetic stability research: Modified UvrB can serve as a tool to study mechanisms of genomic stability in the gut microbiome

To develop such systems:

• Create UvrB variants with enhanced damage recognition capabilities

• Integrate modified uvrB genes into the L. johnsonii genome using CRISPR-Cas9

• Validate functionality through DNA damage response assays

• Test ecological fitness in relevant host environments

What are the common challenges in expressing functional recombinant UvrB in Lactobacillus systems?

Researchers frequently encounter these challenges:

• Low transformation efficiency: L. johnsonii has relatively low transformation rates compared to model organisms

  • Solution: Use glycine-enhanced electroporation protocols and optimize field strength

• Protein solubility issues: UvrB may form inclusion bodies

  • Solution: Express at lower temperatures (25-30°C) and use solubility tags (SUMO, MBP)

• Functional verification: Confirming DNA binding activity

  • Solution: Develop fluorescence-based assays using damaged DNA substrates

• Expression level variations: Inconsistent protein yields

  • Solution: Consider inducible expression systems and optimize media composition

• Contamination with host repair proteins: Co-purification of E. coli UvrB when using E. coli as expression host

  • Solution: Use UvrB-deficient E. coli strains for expression

How can researchers optimize the purification of active recombinant L. johnsonii UvrB protein?

For optimal purification of active UvrB:

• Expression conditions:

  • Use rich media supplemented with 0.2% glucose

  • Induce at OD600 0.6-0.8

  • Express at 30°C for 4-6 hours to balance yield and solubility

• Cell lysis strategy:

  • Use gentle lysis methods (lysozyme treatment followed by sonication)

  • Include DNase I to remove bound DNA that may co-purify with UvrB

  • Maintain reducing conditions with 5mM DTT or β-mercaptoethanol

• Purification protocol:

  • Initial capture: Ni-NTA for His-tagged UvrB

  • Intermediate step: Heparin affinity chromatography to exploit UvrB's DNA-binding properties

  • Polishing: Size exclusion chromatography to separate aggregates

  • Alternative approach: ATP-agarose affinity chromatography exploiting UvrB's ATPase domain

• Activity preservation:

  • Store in buffer containing 10% glycerol, 1mM DTT, and 0.1mM ATP

  • Avoid freeze-thaw cycles; use small aliquots

  • Validate activity using ATPase assays and DNA binding studies

What statistical approaches are most appropriate for analyzing UvrB-DNA binding and repair efficiency data?

For robust statistical analysis of UvrB functional data:

• Binding kinetics:

  • Fit SPR or fluorescence anisotropy data to appropriate binding models (one-site, two-site, cooperative)

  • Use non-linear regression and calculate Ka, Kd values

  • Apply Scatchard analysis to determine binding stoichiometry

• Repair efficiency assays:

  • Use survival curve analysis with log-linear models for UV-sensitivity data

  • Apply ANOVA with post-hoc tests for comparing multiple UvrB variants

  • Consider repeated measures designs when tracking repair over time

• Structure-function correlations:

  • Use multivariate analysis to correlate structural parameters with functional outcomes

  • Apply principal component analysis to identify key structural determinants

  • Consider Bayesian approaches for predictive modeling

• Quality control:

  • Perform power analyses to determine appropriate sample sizes

  • Use bootstrapping for small sample sets

  • Apply appropriate corrections for multiple comparisons (Bonferroni, FDR)

How can researchers resolve contradictory findings when studying L. johnsonii UvrB function across different experimental conditions?

To address contradictory findings:

• Systematic approach to reconciliation:

  • Map differences in experimental conditions (pH, salt concentration, temperature)

  • Compare protein preparation methods for potential activity differences

  • Examine DNA substrate differences (sequence context, lesion type)

• Validate with orthogonal methods:

  • Confirm binding data with multiple techniques (EMSA, fluorescence, SPR)

  • Verify repair activity using complementary assays (in vitro excision, in vivo survival)

  • Use both biochemical and genetic approaches to cross-validate findings

• Consider biological variables:

  • Strain differences in L. johnsonii (as seen in comparative genomic studies)

  • Growth phase effects on UvrB expression and activity

  • Media composition impacts on protein function

• Collaborative verification:

  • Implement standardized protocols across laboratories

  • Share key reagents to eliminate preparation variables

  • Consider round-robin testing of critical findings

When analyzing seemingly contradictory results, examine growth conditions carefully as L. johnsonii strains show significant phenotypic plasticity across environments .

What emerging technologies could advance our understanding of L. johnsonii UvrB function?

Several cutting-edge approaches show promise:

• Cryo-electron microscopy (Cryo-EM): Allows visualization of UvrABC complexes during the repair process at near-atomic resolution

• Single-molecule techniques:

  • DNA curtains to observe UvrB translocation along DNA in real-time

  • Optical tweezers to measure force generation during DNA unwinding

  • smFRET to track conformational changes during damage recognition

• Genomic approaches:

  • ChIP-exo for high-resolution mapping of UvrB binding sites

  • TIME-seq (Transient In vivo Mutational Event sequencing) to map repair events

• Computational methods:

  • Molecular dynamics simulations to model interaction with diverse lesions

  • Machine learning algorithms to predict repair efficiency from DNA sequence context

  • Quantum mechanics/molecular mechanics (QM/MM) to understand the energetics of damage recognition

• Genome editing:

  • CRISPR-Cas9 to create precise UvrB variants in the native L. johnsonii genome

  • Base editors for introducing specific mutations without double-strand breaks

How might the extracellular vesicle production in L. johnsonii affect the study of UvrB function?

The recent discovery of extracellular vesicle (EV) production in L. johnsonii introduces new research considerations:

• Potential presence of UvrB in EVs:

  • UvrB or its fragments might be packaged into EVs, especially under stress conditions

  • EV-associated UvrB could have alternative functions beyond DNA repair

  • May need to purify cellular fractions carefully to avoid EV contamination

• Experimental approaches:

  • Proteomic analysis of L. johnsonii EVs to detect UvrB presence

  • Track UvrB localization under stress conditions that induce EV formation

  • Investigate if DNA damage increases UvrB packaging into EVs

• Biological implications:

  • EV-mediated horizontal transfer of UvrB or uvrB gene fragments between bacteria

  • Potential role in stress response communication within bacterial communities

  • UvrB in EVs might interact with host cells in unique ways

• Methodological considerations:

  • Isolate EVs using differential ultracentrifugation or size exclusion chromatography

  • Use bile (0.2%) to stimulate EV production as recently demonstrated in L. johnsonii N6.2

  • Employ nanoparticle tracking analysis to quantify EVs