Recombinant Vibrio vulnificus DNA ligase (ligA), partial

What is Vibrio vulnificus DNA ligase (ligA) and what is its function in bacterial cells?

Answer: Vibrio vulnificus DNA ligase (LigA) is a NAD-dependent enzyme that catalyzes the formation of phosphodiester linkages between 5'-phosphoryl and 3'-hydroxyl groups in double-stranded DNA . This enzyme (EC 6.5.1.2) is also known as polydeoxyribonucleotide synthase . LigA functions as a critical component in multiple DNA-related processes including:

• Sealing nicks in DNA during replication

• Acting at the final step of recombination by ligating DNA strands

• Participating in various DNA repair pathways

• Contributing to genome stability maintenance

In V. vulnificus specifically, LigA demonstrates high sequence conservation with other bacterial DNA ligases but contains species-specific adaptations that may influence its catalytic efficiency in marine environments where this pathogen resides .

How does V. vulnificus LigA differ structurally from other bacterial DNA ligases?

Answer: V. vulnificus LigA shares the core structural architecture common to NAD-dependent bacterial DNA ligases while exhibiting several distinctive features:

• Domain Organization: Like other bacterial ligases, V. vulnificus LigA contains the conserved nucleotidyltransferase domain with the catalytic lysine residue essential for forming the enzyme-adenylate intermediate .

• Functional Domains: The protein contains several important domains similar to those found in other bacterial ligases:

  • A core nucleotidyltransferase domain

  • An OB-fold domain for DNA binding

  • Specific motifs that dictate NAD+ specificity rather than ATP utilization

• Species-Specific Adaptations: Analysis of V. vulnificus LigA reveals potential selective pressure on certain domains, particularly those involved in DNA binding and catalysis, which may represent adaptations to its specific environmental niche .

V. vulnificus LigA shows approximately 55-60% sequence similarity with DNA ligases from related species such as V. cholerae and V. parahaemolyticus, based on comparative genomic analyses .

What role does V. vulnificus LigA play in homologous recombination pathways?

Answer: V. vulnificus LigA plays a critical role in homologous recombination pathways as evidenced by recent research:

• Position in Recombination Pathways: LigA functions at the terminal step of homologous recombination pathways after the action of RecA, RuvABC complex, or RecG . In V. vulnificus, two general pathways of homologous recombination have been identified:

  • The RecBCD pathway for double-stranded DNA breaks

  • The RecFOR pathway for single-strand breaks

Both ultimately require LigA to seal the processed DNA strands after strand exchange and resolution .

• Interaction with the Holliday Junction Resolvasome: Research indicates that LigA works in concert with the Holliday junction resolution machinery (RuvABC) to complete recombination events . The RuvABC complex includes:

  • RuvA: Binds to DNA and senses structure

  • RuvB: Provides ATP-driven energy for junction migration

  • RuvC: Acts as an endonuclease to resolve the junction

  • LigA: Seals the resulting nicks after resolution

• Specialization Score Analysis: Comparative genomic studies have assigned LigA a high "specialization score" (SS = 0.77) in certain bacterial species, suggesting its importance in enhanced recombination capacity . This places it alongside key recombination proteins like RuvA (SS = 0.88), RuvB (SS = 0.84), and RuvC (SS = 0.73).

What experimental approaches can be used to assess the activity of recombinant V. vulnificus LigA?

Answer: Several experimental approaches can be employed to study recombinant V. vulnificus LigA activity:

• In vitro Ligation Assay:

  • Prepare nicked DNA substrates using restriction enzymes or DNase I under controlled conditions

  • Incubate with purified recombinant LigA in presence of NAD+ and Mg2+

  • Analyze ligation products via gel electrophoresis

  • Quantify the percentage of sealed DNA to determine activity

• Adenylylation Assay:

  • Measure the formation of the enzyme-adenylate intermediate using [α-32P]NAD+

  • Analyze by SDS-PAGE and autoradiography to detect labeled enzyme

  • This method can determine the efficiency of the first step in the ligation reaction

• Thermostability Assessment:

  • Test LigA activity at different temperatures (20-90°C) to determine optimal conditions

  • V. vulnificus LigA may exhibit different thermal properties compared to other bacterial ligases due to its marine environment adaptation

  • Based on studies of thermophilic ligases, assays at 70-90°C can be used as a comparison benchmark

• Nick-Sealing Kinetics:

  • Monitor the rate of nick-sealing under different conditions (pH, salt concentration, temperature)

  • Compare kinetic parameters (KM, kcat) with other bacterial ligases

  • Determine the rate-limiting step in the ligation process

• Functional Complementation:

  • Express V. vulnificus ligA in E. coli ligA temperature-sensitive mutants

  • Assess growth restoration at non-permissive temperatures

  • This approach was successfully used for studying DNA ligases from other species and can be applied to V. vulnificus LigA

How does LigA contribute to V. vulnificus virulence and survival in different environments?

Answer: Research suggests LigA plays complex roles in V. vulnificus virulence and environmental adaptation:

• DNA Damage Repair in Host Environments:

  • V. vulnificus encounters oxidative stress within host cells that causes DNA damage

  • LigA is essential for repairing damage to maintain genomic integrity during infection

  • Enhanced DNA repair capability may contribute to bacterial persistence in host environments

• Serum Resistance Connection:

  • V. vulnificus strains with intact DNA repair pathways show better survival in human serum

  • While direct evidence for LigA's role in serum resistance is limited, genomic studies have identified several genes contributing to serum resistance in V. vulnificus

  • LigA likely supports genomic stability under serum stress conditions

• Temperature Adaptation:

  • V. vulnificus must adapt to temperature shifts between marine environments (~20°C) and human hosts (37°C)

  • LigA activity may be modulated to maintain function across this temperature range

  • This adaptation is crucial for the pathogen's lifecycle

• Link to Mortality:

  • Clinical studies have correlated V. vulnificus DNA load with mortality in infected patients

  • Higher bacterial loads (measured by real-time PCR) were significantly associated with fatal outcomes

  • This suggests that bacteria with intact replication and repair machinery (including functional LigA) may achieve higher in vivo loads

The relationship between specific LigA variants and V. vulnificus virulence remains an area requiring further research, as noted in genomic studies of clinical isolates that show genetic divergence without clear correlation to disease severity .

What are the optimal conditions for expressing and purifying recombinant V. vulnificus LigA?

Answer: Based on current protocols for recombinant V. vulnificus proteins, the following optimized workflow is recommended:

• Expression System:

  • Host: E. coli BL21(DE3) or similar expression strain

  • Vector: pET system with T7 promoter control

  • Fusion tags: N-terminal His6 tag for purification or dual affinity tags for enhanced purity

• Expression Conditions:

  • Culture medium: LB or 2×YT supplemented with appropriate antibiotics

  • Induction: IPTG (0.1-0.5 mM) at OD600 of 0.6-0.8

  • Temperature: 16-18°C for overnight expression to enhance solubility

  • This slow expression approach is critical for obtaining enzymatically active LigA

• Cell Lysis and Initial Purification:

  • Buffer composition: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 0.1% Triton X-100

  • Lysis method: Sonication or French press

  • Clarification: Centrifugation at 20,000 × g for 30 min at 4°C

• Purification Strategy:

  • Initial capture: Ni-NTA affinity chromatography

  • Intermediate purification: Ion exchange chromatography (HiTrap Q)

  • Polishing: Size exclusion chromatography (Superdex 200)

  • Buffer exchange to storage buffer: 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 50% glycerol

• Quality Control:

  • Purity assessment: SDS-PAGE (target >85% purity)

  • Activity verification: Nick-sealing assay using synthetic nicked DNA substrates

  • Storage: Aliquot and store at -80°C to maintain activity

The choice of E. coli as an expression system is supported by successful production of other V. vulnificus recombinant proteins, including the partial LigA product described in commercial sources .

How can researchers assess the function of LigA in V. vulnificus DNA repair pathways?

Answer: Multiple complementary approaches can be employed to investigate LigA's role in V. vulnificus DNA repair:

• Construction of Genomic Mutants:

  • Create a V. vulnificus vvpD mutant through allelic exchange, as demonstrated for other V. vulnificus genes

  • Generate a LigA-deficient strain using suicide vectors like pEP185.2 carrying a disrupted ligA gene

  • Create complemented strains by introducing intact ligA on a plasmid vector

  • Use tools similar to those described for V. vulnificus vvn gene studies

• DNA Damage Response Assays:

  • Expose wild-type and LigA-modified strains to DNA-damaging agents:

    • UV irradiation (254 nm)

    • Hydrogen peroxide (oxidative damage)

    • Mitomycin C (crosslinking agent)

  • Compare survival rates and recovery kinetics

  • Measure mutation frequencies using rifampicin resistance assays

• Recombination Frequency Measurement:

  • Adapt conjugation and transformation protocols to assess recombination efficiency

  • Based on V. vulnificus nuclease studies, transformation frequencies can be measured under controlled conditions

  • Compare recombination frequencies between wild-type and LigA-variant strains

• Protein-Protein Interaction Studies:

  • Investigate LigA's interaction with other DNA repair proteins (RuvABC, RecG)

  • Use bacterial two-hybrid assays or co-immunoprecipitation

  • Map interaction domains through truncation or site-directed mutagenesis

• In vivo DNA Repair Kinetics:

  • Monitor repair of induced DNA breaks using pulse-field gel electrophoresis

  • Compare kinetics between wild-type and LigA-modified strains

  • Quantify DNA damage resolution over time

This multi-faceted approach allows comprehensive characterization of LigA's role in maintaining genomic integrity under various stress conditions encountered by V. vulnificus.

What techniques can be used to study the structure-function relationship of V. vulnificus LigA domains?

Answer: Several advanced techniques can elucidate the structure-function relationship of V. vulnificus LigA:

• Domain Mapping and Mutagenesis:

  • Generate truncated versions of LigA to identify minimal functional domains

  • Create single amino acid substitutions at conserved residues based on sequence alignment with other bacterial ligases

  • Focus on the catalytic lysine residue essential for enzyme-adenylate formation

  • Target residues in the BRCA1 C-terminal domain that show high dN/dS ratios indicating selective pressure

• Structural Analysis:

  • X-ray crystallography of purified LigA (both full-length and individual domains)

  • Cryo-EM analysis of LigA in complex with nicked DNA substrates

  • NMR spectroscopy for studying dynamics of smaller domains

  • Homology modeling based on solved structures of related bacterial DNA ligases

• Functional Domain Analysis:

• Comparative Approach:

  • Utilize knowledge from RuvABC complex structural studies as a framework

  • Compare with LigA from other Vibrio species to identify V. vulnificus-specific features

  • Analyze domains under positive selection as identified in comparative genomic studies

• Biophysical Characterization:

  • Circular dichroism to analyze secondary structure

  • Thermal shift assays to determine domain stability

  • Fluorescence spectroscopy to monitor conformational changes during catalysis

  • Surface plasmon resonance to quantify DNA binding kinetics

These approaches will provide insights into how the structural features of V. vulnificus LigA contribute to its function in the context of bacterial DNA repair and recombination.

How might studies of V. vulnificus LigA contribute to understanding bacterial pathogenesis?

Answer: Research on V. vulnificus LigA has several important implications for understanding bacterial pathogenesis:

• Genomic Plasticity and Adaptation:

  • V. vulnificus shows substantial genome plasticity, with strain-specific genomic islands

  • LigA may facilitate genomic rearrangements through its role in recombination

  • Understanding LigA's contribution to genomic plasticity could explain adaptation to different hosts and environments

• Survival Under Host-Induced Stress:

  • During infection, V. vulnificus faces oxidative stress from host immune responses

  • LigA-mediated DNA repair likely contributes to bacterial persistence

  • Comparative analysis of LigA activity in clinical versus environmental isolates could reveal adaptations for virulence

• Virulence-Associated Genomic Islands:

  • Studying LigA's role in maintaining and integrating genomic islands containing virulence factors

  • Research suggests V. vulnificus pathogenicity islands show high levels of genome plasticity

  • LigA may participate in horizontal gene transfer events that enhance virulence

• Biofilm Formation and Persistence:

  • DNA repair mechanisms likely contribute to biofilm formation and stability

  • LigA's role in maintaining genomic integrity within biofilms remains largely unexplored

  • Connection to two-component systems and quorum sensing pathways identified in genomic studies

• Evolution of Virulence:

  • Phylogenetic analyses show V. vulnificus strains fall into distinct lineages with varying virulence

  • LigA variants may contribute to lineage-specific characteristics through effects on genomic stability

  • Genetic variability facilitated by recombination systems could drive the emergence of hypervirulent strains

What are the promising research directions for studying V. vulnificus LigA in the context of bacterial genetics?

Answer: Several promising research directions emerge from current understanding of V. vulnificus LigA:

• Comparative Genomics Approach:

  • Analyze LigA sequence variation across clinical and environmental V. vulnificus isolates

  • Correlate specific LigA variants with virulence phenotypes or environmental adaptations

  • Expand current multilocus sequence typing schemes to include ligA for enhanced strain discrimination

• Gene Editing Applications:

  • Explore LigA's potential utility in CRISPR-Cas9-assisted genetic engineering

  • Investigate how V. vulnificus LigA might function in non-homologous end-joining (NHEJ) or alternative end-joining (A-EJ) pathways

  • Develop improved genetic manipulation tools for Vibrio species based on LigA properties

• Environmental Adaptation Studies:

  • Investigate how LigA function varies under different environmental conditions (temperature, salinity, pH)

  • Determine if LigA shows differential activity in clinical versus environmental settings

  • Study the role of LigA in horizontal gene transfer events in marine environments

• Systems Biology Integration:

  • Incorporate LigA into genome-scale metabolic models of V. vulnificus

  • Map interactions between DNA repair pathways and other cellular processes

  • Identify potential synthetic lethal interactions involving ligA for antimicrobial development

• Evolutionary Studies:

  • Analyze selection pressure on ligA across Vibrio species to identify adaptive changes

  • Investigate the evolution of NAD+-dependent ligases versus ATP-dependent variants

  • Study the co-evolution of LigA with other components of the DNA repair machinery

These research directions promise to advance our understanding of bacterial genetics, pathogenesis, and potential applications in biotechnology.

What are the common challenges in working with recombinant V. vulnificus LigA and how can they be addressed?

Answer: Researchers working with recombinant V. vulnificus LigA face several technical challenges:

• Protein Solubility Issues:

  • Challenge: LigA often forms inclusion bodies during heterologous expression

  • Solution:

    • Express at lower temperatures (16-18°C)

    • Use solubility-enhancing fusion tags (SUMO, MBP)

    • Optimize induction conditions (lower IPTG concentration, 0.1-0.2 mM)

    • Consider specialized E. coli strains like Rosetta(DE3) to address potential codon bias

• Activity Loss During Purification:

  • Challenge: LigA is sensitive to oxidation, affecting catalytic activity

  • Solution:

    • Include reducing agents (DTT or β-mercaptoethanol) in all buffers

    • Add NAD+ (0.1-0.5 mM) in purification buffers to stabilize the enzyme

    • Work rapidly and avoid freeze-thaw cycles

    • Note that LigA is active in oxidized form but not reduced form for nuclease activity

• Substrate Specificity Determination:

  • Challenge: Distinguishing between DNA vs. RNA ligation activity

  • Solution:

    • Perform differential thermal stability assays (DNase activity is more thermostable than RNase activity)

    • Design specific substrates with unique secondary structures

    • Conduct side-by-side comparison with known RNA and DNA ligases

• Validation in V. vulnificus Systems:

  • Challenge: Limited genetic tools for V. vulnificus compared to E. coli

  • Solution:

    • Adapt methods from successful V. vulnificus genetic studies

    • Develop complementation systems in V. vulnificus LigA mutants

    • Use suicide vectors like pCVD442 with sacB for selection

• Storage Stability:

  • Challenge: Activity loss during storage

  • Solution:

    • Store in buffer containing 25-50% glycerol at -80°C

    • Aliquot to avoid repeated freeze-thaw cycles

    • Consider lyophilization for long-term storage (12 months at -20°C)

    • Add stabilizing agents like BSA (0.1 mg/ml) to diluted working stocks

How can researchers effectively compare V. vulnificus LigA activity with other bacterial DNA ligases?

Answer: Standardized comparative analyses require careful experimental design:

• Standardized Activity Assays:

  • Establish consistent substrate preparation methods:

    • Prepare identical nicked DNA substrates for all ligases

    • Use defined oligonucleotide sequences with standardized fluorescent or radioactive labels

  • Measure activity under multiple conditions:

    • Temperature range (20-90°C)

    • pH range (6.0-9.0)

    • Salt concentration variations (50-500 mM NaCl)

• Kinetic Parameter Determination:

  • Measure and compare fundamental kinetic parameters:

  Parameter	Measurement Method	Typical Range for Bacterial Ligases
  kcat	Initial velocity measurements at saturating substrate	0.01-10 s⁻¹
  KM (NAD+)	Vary NAD+ concentration at fixed DNA	0.1-10 μM
  KM (nicked DNA)	Vary DNA concentration at fixed NAD+	5-100 nM
  Thermal stability	Activity retention after heat treatment	Species-dependent

• Substrate Preference Analysis:

  • Test activity on various DNA structures:

    • Cohesive-end substrates

    • Blunt-end substrates

    • Single-strand breaks with various gap sizes

    • RNA-DNA hybrid substrates

• Cofactor Specificity:

  • Determine absolute requirement for NAD+ vs. ATP

  • Test activity in presence of different divalent cations (Mg²⁺, Mn²⁺, Ca²⁺)

  • Compare with ATP-dependent ligases from other organisms

• Structural Comparison:

  • Align sequences to identify conserved and divergent regions

  • Compare domain organization

  • Analyze structure-based evolutionary relationships

  • Focus analysis on the five conserved peptide motifs in the nucleotidyltransferase domain