Recombinant Pseudomonas syringae pv. tomato DNA ligase B (ligB), partial

What is DNA ligase B (ligB) from Pseudomonas syringae pv. tomato?

DNA ligase B (ligB) from Pseudomonas syringae pv. tomato is an NAD-dependent DNA ligase that catalyzes the formation of phosphodiester linkages between 5'-phosphoryl and 3'-hydroxyl groups in double-stranded DNA. This enzyme, also known as polydeoxyribonucleotide synthase [NAD(+)] B, uses NAD as both a coenzyme and energy source for the reaction. The recombinant form has a molecular weight of approximately 62,020 Da and belongs to the DNA ligase protein family. LigB plays essential roles in DNA replication, repair, and recombination processes in P. syringae pv. tomato .

How does ligB differ structurally and functionally from other DNA ligases?

LigB from P. syringae pv. tomato differs from other DNA ligases primarily in its cofactor requirements and domain architecture. While eukaryotic DNA ligases typically use ATP as a cofactor, ligB is NAD-dependent, a characteristic common to bacterial DNA ligases. The protein contains conserved motifs for NAD+ binding and DNA interaction, but exhibits sequence variations that may affect substrate specificity and catalytic efficiency compared to other bacterial ligases. Functionally, ligB is involved in multiple DNA repair pathways including base excision repair, nucleotide excision repair, and mismatch repair, in addition to DNA replication . These multiple pathway involvements suggest a versatile role in maintaining genomic integrity in P. syringae, potentially differentiating it from more specialized ligases in other organisms.

What are the conserved domains and active sites in ligB that are critical for its function?

The ligB protein contains several conserved domains crucial for its enzymatic activity. The N-terminal domain houses the NAD+-binding pocket characterized by a Rossmann fold, while the central domain contains the adenylation site where the AMP moiety is transferred to a conserved lysine residue during catalysis. The C-terminal domain is responsible for DNA binding and positioning. Critical active site residues include the catalytic lysine that forms the covalent enzyme-AMP intermediate, acidic residues that coordinate metal ions necessary for phosphodiester bond formation, and basic residues that interact with the DNA backbone. Mutations in these conserved regions typically result in significant reduction or complete loss of enzymatic activity, highlighting their essentiality for ligB function .

What are the optimal expression systems for producing recombinant ligB?

The optimal expression systems for producing recombinant ligB from P. syringae pv. tomato include E. coli, yeast, baculovirus, and mammalian cell systems, with E. coli being the most commonly used for its simplicity and high yield . For E. coli expression, BL21(DE3) or similar strains are recommended due to their reduced protease activity. Expression constructs typically incorporate an N-terminal tag (such as His6 or GST) to facilitate purification, and expression is often induced at lower temperatures (16-25°C) to improve protein solubility. Yeast systems like Pichia pastoris may offer advantages for proteins requiring post-translational modifications, while baculovirus and mammalian cell systems provide more complex eukaryotic processing machinery but at higher cost and complexity. The choice between these systems should be guided by the specific research requirements, particularly regarding protein folding, modification needs, and downstream applications .

What purification strategies yield the highest purity and activity for recombinant ligB?

High-purity, active recombinant ligB can be achieved through a multi-step purification strategy. Initially, affinity chromatography utilizing the protein's N-terminal tag (typically His6) allows for selective binding to nickel or cobalt resins. Following elution with imidazole, ion exchange chromatography (typically using a Q-Sepharose column at pH 8.0) helps remove contaminating proteins with different charge characteristics. Size exclusion chromatography serves as a polishing step to separate ligB from aggregates and differently sized impurities. Throughout purification, maintaining buffer conditions with 10-20% glycerol, 1-5 mM DTT or β-mercaptoethanol, and 0.1-0.5 M NaCl helps preserve protein stability and activity. The final product typically achieves ≥85% purity as determined by SDS-PAGE . For applications requiring extremely high purity, additional steps such as hydroxyapatite chromatography may be incorporated. Activity assessments using NAD+-dependent DNA ligation assays should be performed after each purification step to monitor retention of enzymatic function.

How should researchers approach tag selection and removal for ligB studies?

When selecting tags for recombinant ligB expression and purification, researchers should consider both experimental objectives and the protein's biochemical properties. The recombinant ligB typically contains an N-terminal tag, with potential for a C-terminal tag as well . For structural studies, smaller tags like His6 minimize interference with protein folding, while larger tags like MBP or GST may enhance solubility but require removal before crystallization attempts. For interaction studies, fluorescent protein tags enable visualization but may alter binding kinetics.

For tag removal, site-specific proteases (TEV, PreScission, or thrombin) can be employed, with cleavage sites engineered between the tag and ligB sequence. Optimization of cleavage conditions is essential, typically using 1:50-1:100 protease:ligB ratio at 4°C overnight in buffers containing 20-50 mM Tris-HCl (pH 7.5-8.0), 100-200 mM NaCl, and 1-2 mM DTT. Post-cleavage, reversed affinity chromatography removes the cleaved tag and uncleaved protein, while gel filtration ensures homogeneity. Researchers should verify complete tag removal via Western blotting or mass spectrometry and confirm that enzyme activity is maintained or enhanced after tag removal through ligase activity assays .

What assays are most effective for measuring ligB enzymatic activity?

The most effective assays for measuring ligB enzymatic activity include:

• Gel-based DNA ligation assay: This method uses 5'-phosphorylated, nicked double-stranded DNA substrates, typically with a fluorescent label. After incubation with ligB and NAD+, reaction products are analyzed by gel electrophoresis, with ligated products migrating differently than substrates. Quantification via densitometry or fluorescence scanning provides activity measurements.

• Real-time fluorescence assays: These utilize specially designed DNA substrates with fluorophore-quencher pairs that change fluorescence properties upon ligation, allowing continuous monitoring of activity.

• Radioactive assays: Using 32P-labeled DNA substrates offers high sensitivity for detecting low levels of ligation activity.

• Coupled enzyme assays: These monitor NAD+ consumption or nicotinamide production through secondary enzymatic reactions with spectrophotometric readouts.

Optimal reaction conditions typically include 50 mM Tris-HCl (pH 7.5-8.0), 5-10 mM MgCl2, 1-5 mM DTT, 50-100 µg/ml BSA, 0.5-1 mM NAD+, and 0.1-1 µM DNA substrate at 30-37°C . These assays can be adapted to determine kinetic parameters (KM, kcat), investigate substrate preferences, or screen potential inhibitors.

How does ligB participate in DNA repair and recombination pathways?

DNA ligase B from P. syringae pv. tomato participates in multiple DNA repair and recombination pathways, serving as a critical enzyme for maintaining genomic integrity. In base excision repair, ligB seals nicks created after damaged base removal and DNA synthesis. During nucleotide excision repair, it completes the final ligation step after damaged nucleotide removal and gap filling . In mismatch repair, ligB restores strand continuity following removal of mismatched nucleotides. Additionally, the enzyme plays a role in DNA replication by joining Okazaki fragments on the lagging strand.

In recombination processes, ligB works in concert with recombineering proteins like RecE and RecT homologs identified in P. syringae . These systems facilitate homologous recombination between genomic loci and linear DNA substrates introduced into cells, enabling genetic engineering applications. The RecE/RecT-like proteins promote efficient recombination, with RecT alone sufficient for single-stranded DNA oligonucleotide recombination, while both RecE and RecT are required for double-stranded DNA recombination . LigB likely functions in these pathways by completing the recombination process through ligation of DNA strands, working synergistically with the recombination machinery to maintain chromosome integrity.

How can ligB be utilized in recombineering systems for P. syringae?

LigB can be strategically incorporated into recombineering systems for P. syringae to enhance the efficiency of genetic manipulation. When combined with recombination proteins like RecE and RecT homologs identified in P. syringae pv. syringae B728a, ligB can significantly improve the integration of linear DNA fragments into the bacterial chromosome . This integration approach requires:

• Expression optimization: Co-expressing ligB with recombination proteins, ideally under the control of an inducible promoter to prevent potential toxicity from overexpression.

• Substrate design: Creating linear DNA substrates with homology arms (40-50 bp) flanking the desired modification, with phosphorylated 5' ends to facilitate ligB-mediated joining.

• Delivery methodology: Introducing the DNA substrate via electroporation using optimized parameters (e.g., 2.5 kV, 200 Ω, 25 μF) in competent cells expressing the recombineering machinery.

This system enables various genetic modifications including gene deletions, insertions, and point mutations with significantly higher efficiency than traditional methods. For single-stranded DNA recombination, RecT alone is sufficient, while efficient double-stranded DNA recombination requires both RecT and RecE expression . LigB contributes to this process by sealing remaining nicks after recombination, potentially increasing the stability of the integrated DNA and reducing the likelihood of degradation by exonucleases.

What advantages does ligB offer over other DNA ligases for bacterial genome editing?

LigB from P. syringae pv. tomato offers several distinct advantages for bacterial genome editing compared to other DNA ligases:

• Native compatibility: Being derived from Pseudomonas, ligB likely functions optimally under the physiological conditions present in pseudomonad cells, potentially offering higher activity in these bacterial systems compared to ligases from distantly related organisms .

• NAD+ dependency: Unlike ATP-dependent ligases, ligB utilizes NAD+ as a cofactor, which is typically more abundant in bacterial cells, potentially providing more consistent activity during editing procedures .

• Coordination with recombination machinery: When used alongside native recombineering proteins like RecE and RecT homologs from P. syringae, ligB likely has evolved for optimal interaction with these components, potentially enhancing the efficiency of the complete recombination process .

• Temperature flexibility: LigB maintains activity across a broader temperature range than some other ligases, allowing protocols to be conducted at lower temperatures (20-30°C) that may better preserve the integrity of sensitive DNA constructs.

• Reduced immunogenicity: For applications in P. syringae, using the native ligase minimizes the introduction of foreign proteins that might trigger bacterial defense mechanisms that could reduce editing efficiency.

• Pathway integration: LigB naturally participates in multiple DNA repair pathways in Pseudomonas, potentially allowing edited regions to be more efficiently processed through these endogenous systems, reducing unintended mutations or rearrangements .

These advantages make ligB particularly valuable for precision engineering of Pseudomonas genomes, offering improvements over heterologous systems that rely on ligases from E. coli or other organisms.

How does ligB compare to λ Red recombineering systems for Pseudomonas modification?

LigB-based systems and λ Red recombineering represent distinct approaches to Pseudomonas genome modification, each with specific advantages and limitations:

Methodological differences:

• Component requirements: λ Red systems require expression of three phage genes (gam, bet, exo) in recipient Pseudomonas cells , whereas ligB-based approaches can leverage native Pseudomonas recombination machinery (RecE/RecT homologs) complemented with ligB .

• Substrate preparation: λ Red systems for P. aeruginosa require very long primers or multi-fragment splicing-by-overlap-extension (SOE)-PCR to generate mutant alleles , while ligB-based systems can often use simpler substrate designs.

• Recipient strain preparation: λ Red recombination requires an initial transformation step to introduce vectors expressing the recombination genes, whereas two-step allelic exchange utilizing native machinery with ligB can be applied directly to many genetic backgrounds of Pseudomonas .

Performance comparison:

• Efficiency: Native ligB working with Pseudomonas RecE/RecT homologs can achieve higher recombination frequencies in Pseudomonas strains compared to heterologous λ Red systems, as the native proteins are evolutionarily adapted to function optimally in pseudomonad cellular environments .

• Versatility: Two-step allelic exchange approaches incorporating ligB allow for introducing the desired mutation into many strains in parallel or creating multiple mutations in a single strain sequentially, without requiring additional transformations to create suitable recipient strains .

• Precision: Both systems can achieve single-nucleotide precision, but ligB-based systems potentially offer advantages in maintaining DNA integrity during recombination due to optimized coordination between the native ligase and recombination machinery .

For Pseudomonas-specific applications, ligB-based systems represent an increasingly preferred approach due to their streamlined methodology and enhanced compatibility with the native cellular environment.

How can researchers develop inhibitors targeting ligB for antimicrobial applications?

Developing inhibitors targeting ligB from P. syringae pv. tomato for antimicrobial applications requires a systematic approach combining structural analysis, biochemical screening, and validation studies:

• Structure-based design strategy:

  • Utilize X-ray crystallography or homology modeling to identify ligB's active site architecture

  • Focus on the NAD+-binding pocket and DNA-interaction domains as primary targets

  • Employ molecular docking to screen virtual compound libraries against these sites

  • Prioritize compounds that interact with catalytic residues or that disrupt cofactor binding

• Biochemical screening approach:

  • Develop a high-throughput ligase activity assay using fluorescence detection

  • Screen chemical libraries for compounds that inhibit ligB activity

  • Establish dose-response relationships to determine IC50 values

  • Perform kinetic analyses to determine inhibition mechanisms (competitive, noncompetitive, etc.)

• Selectivity assessment:

  • Test promising inhibitors against human DNA ligases to ensure selectivity

  • Evaluate activity against DNA ligases from beneficial bacteria to minimize microbiome disruption

  • Quantify selectivity indices to prioritize compounds with favorable therapeutic windows

• Antimicrobial validation:

  • Determine minimum inhibitory concentrations against P. syringae cultures

  • Assess inhibitor stability and penetration into bacterial cells

  • Evaluate potential for resistance development through serial passage experiments

  • Test efficacy in plant infection models to confirm in vivo activity

This approach mirrors successful strategies used for developing inhibitors against virulence factors from other pathogens, such as the LasB elastase from P. aeruginosa, where recombinant protein production enabled accelerated drug development .

What methodologies can elucidate the structural dynamics of ligB during catalysis?

Elucidating the structural dynamics of ligB during catalysis requires a multi-technique approach that captures the enzyme's conformational changes throughout the reaction cycle:

• Time-resolved X-ray crystallography:

  • Trap ligB in various catalytic states using substrate analogs or non-hydrolyzable NAD+ derivatives

  • Employ microcrystal techniques with synchrotron radiation or X-ray free-electron lasers

  • Analyze structures sequentially to map conformational transitions during adenylation and ligation

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Monitor solvent accessibility changes in different regions of ligB upon substrate/cofactor binding

  • Compare deuterium incorporation patterns with and without DNA substrates or NAD+

  • Identify flexible regions that undergo significant conformational rearrangements

• Single-molecule FRET spectroscopy:

  • Design ligB constructs with strategically placed fluorophore pairs

  • Monitor real-time distance changes between protein domains during catalysis

  • Correlate FRET efficiency changes with specific steps in the reaction mechanism

• Molecular dynamics simulations:

  • Build atomistic models based on crystal structures

  • Simulate microsecond-scale dynamics during substrate binding and catalysis

  • Identify transient interactions and water networks critical for catalysis

• NMR spectroscopy:

  • Perform 15N/13C-labeling of recombinant ligB

  • Analyze chemical shift perturbations upon ligand binding

  • Study relaxation dispersion to identify residues involved in microsecond-millisecond conformational exchange

These complementary approaches would provide unprecedented insights into ligB's catalytic mechanism, revealing how domain movements and active site rearrangements contribute to its NAD+-dependent DNA ligation activity .

How might ligB be engineered for enhanced activity or altered specificity?

Engineering ligB for enhanced activity or altered specificity can be approached through several protein engineering strategies:

• Rational design based on structural knowledge:

  • Identify rate-limiting steps through kinetic analysis

  • Modify residues in the active site to enhance NAD+ binding or adenylation efficiency

  • Engineer the DNA-binding domain to improve substrate recognition

  • Introduce stabilizing interactions to increase thermostability without compromising flexibility

• Directed evolution approaches:

  • Develop a high-throughput screening system based on bacterial survival under selection pressure

  • Create libraries through error-prone PCR or DNA shuffling with homologous ligases

  • Perform multiple rounds of selection to identify variants with desired properties

  • Combine beneficial mutations to achieve additive or synergistic effects

• Domain swapping and chimeric enzymes:

  • Replace specific domains with counterparts from ligases with desired properties

  • Create chimeras with ATP-dependent ligases to develop dual-cofactor enzymes

  • Engineer hybrid DNA-binding domains to alter substrate specificity

  • Incorporate domains from thermophilic ligases to enhance stability

• Computationally guided approaches:

  • Use machine learning to predict mutations that enhance specific properties

  • Apply molecular dynamics to identify dynamic bottlenecks in catalysis

  • Design stabilizing networks of hydrogen bonds or salt bridges

  • Simulate protein-DNA interactions to guide specificity alterations

• Post-translational modification engineering:

  • Introduce sites for controlled phosphorylation to regulate activity

  • Modify surface residues to alter solubility or cellular localization

  • Engineer cysteines for reversible redox regulation

These engineering strategies could yield ligB variants with applications in biotechnology, synthetic biology, and improved recombineering systems . Successful engineering might produce variants with enhanced ligation efficiency at lower temperatures, broader substrate scope, or altered cofactor preferences.

What are common challenges in expressing active ligB and how can they be overcome?

Researchers commonly encounter several challenges when expressing active ligB from P. syringae pv. tomato, each requiring specific troubleshooting approaches:

• Protein insolubility:

  • Problem: Formation of inclusion bodies due to misfolding

  • Solutions:

    • Lower induction temperature to 16-20°C

    • Reduce inducer concentration (0.1-0.25 mM IPTG)

    • Co-express with chaperones (GroEL/ES, DnaK/J)

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

    • Add stabilizing additives to lysis buffer (10% glycerol, 0.1% Triton X-100)

• Low enzymatic activity:

  • Problem: Properly folded protein with suboptimal activity

  • Solutions:

    • Ensure preservation of reducing environment (5 mM DTT in buffers)

    • Add divalent cations (MgCl₂, MnCl₂) to stabilize active site

    • Optimize protein extraction conditions to minimize proteolysis

    • Test multiple expression constructs with different tag positions

    • Verify NAD⁺ quality and concentration in activity assays

• Proteolytic degradation:

  • Problem: Enzyme fragmentation during expression or purification

  • Solutions:

    • Use protease-deficient strains (BL21(DE3), Rosetta)

    • Include protease inhibitors in all buffers

    • Maintain low temperatures during purification

    • Minimize time between cell disruption and purification

    • Consider on-column refolding techniques

• Cofactor binding issues:

  • Problem: Recombinant ligB exhibits reduced NAD⁺ binding

  • Solutions:

    • Include low concentrations of NAD⁺ (0.1-0.5 mM) in purification buffers

    • Analyze protein by thermal shift assay to optimize stabilizing conditions

    • Verify correct folding of the NAD⁺-binding domain through circular dichroism

    • Test structurally validated buffer conditions that maintain NAD⁺-dependent enzymes

By systematically addressing these challenges, researchers can significantly improve yields of active ligB for downstream applications in DNA manipulation, bacterial genome engineering, and biochemical characterization studies .

How can researchers optimize ligB activity assays for different experimental contexts?

Optimizing ligB activity assays for different experimental contexts requires tailoring approaches based on specific research objectives:

• High-throughput inhibitor screening:

  • Use fluorescence-based microplate assays with hairpin substrates containing fluorophore-quencher pairs

  • Optimize signal-to-noise ratio through substrate concentration adjustments (typically 50-200 nM)

  • Develop Z-factor analysis to ensure statistical robustness (aim for Z > 0.7)

  • Include positive controls (known inhibitors) and negative controls (buffer only)

  • Prepare master mixes to minimize pipetting steps and reduce variability

• Detailed kinetic characterization:

  • Employ radiometric assays using ³²P-labeled substrates for highest sensitivity

  • Design time-course experiments with multiple substrate concentrations

  • Maintain enzyme concentration below 10% of substrate to ensure steady-state conditions

  • Include controls for background adenylation and non-enzymatic ligation

  • Analyze data using appropriate kinetic models (Michaelis-Menten, Hill equation)

• Specificity determination:

  • Prepare DNA substrates with systematic variations (mismatches, gaps, different sequences)

  • Normalize activity measurements to standard substrate control

  • Use gel-based assays for direct visualization of ligation products

  • Implement competition assays between different substrates

  • Analyze results using relative efficiency calculations

• In vitro recombination applications:

  • Optimize buffer conditions to enable coupling with recombination proteins (RecE/RecT)

  • Adjust NAD⁺ concentration (0.5-2 mM) for sustained activity in longer reactions

  • Include molecular crowding agents (PEG-8000) to mimic cellular environment

  • Control reaction temperature (25-30°C) to balance enzyme activity and substrate stability

  • Verify complete reactions by transformation efficiency assessment

• Structure-function analysis:

  • Design partial reaction assays to isolate adenylation and nick-sealing steps

  • Use non-hydrolyzable NAD⁺ analogs to trap reaction intermediates

  • Incorporate spectroscopic probes to monitor conformational changes

  • Employ site-directed mutants as controls for specific catalytic steps

  • Analyze data against structural models to correlate activity with protein dynamics

These optimized approaches enable researchers to extract maximum information from ligB activity assays, supporting applications ranging from basic mechanistic studies to applied genetic engineering contexts .

What strategies can address problems with ligB stability during purification and storage?

To address stability challenges with ligB during purification and storage, researchers can implement several strategies that preserve both structural integrity and enzymatic activity:

• Buffer optimization during purification:

  • Include stabilizing additives: 10-20% glycerol to prevent aggregation, 1-5 mM DTT to maintain reduced cysteines

  • Optimize ionic strength: 150-300 mM NaCl to shield electrostatic interactions

  • Maintain optimal pH range: typically 7.5-8.0 to prevent acid-catalyzed degradation

  • Add low concentrations of substrate-mimicking compounds or cofactors (0.1 mM NAD+)

  • Consider surfactants at sub-CMC concentrations (0.01-0.05% Triton X-100) to prevent adsorption losses

• Purification procedure modifications:

  • Minimize time between purification steps to reduce exposure to proteases

  • Maintain low temperature (4°C) throughout all procedures

  • Consider on-column refolding for proteins recovered from inclusion bodies

  • Use gradient elution profiles to separate correctly folded protein from aggregates

  • Employ size exclusion chromatography as a final polishing step to ensure homogeneity

• Long-term storage approaches:

  • Store at -80°C in small (50-100 μL) single-use aliquots to prevent freeze-thaw damage

  • For lyophilized preparations, include cryoprotectants like trehalose or sucrose (5-10%)

  • Test stability with various excipients (amino acids, polyols) to identify optimal formulations

  • Consider flash-freezing in liquid nitrogen rather than slow freezing

  • Validate activity retention after various storage periods (1 week, 1 month, 3 months)

• Protein engineering solutions:

  • Identify and mutate surface-exposed cysteine residues to serine to prevent disulfide-mediated aggregation

  • Consider rational design of stabilizing salt bridges or hydrophobic interactions

  • Remove protease-sensitive loops if structural information is available

  • Explore fusion partners known to enhance stability (MBP, thioredoxin)

• Quality control protocols:

  • Implement thermal shift assays (Thermofluor) to monitor stability improvements

  • Use dynamic light scattering to detect early signs of aggregation

  • Develop activity assays that can be performed quickly to verify functional integrity

  • Monitor protein by SDS-PAGE before and after storage to assess degradation

These comprehensive strategies can significantly improve ligB stability, ensuring that researchers work with functional protein throughout their experimental workflows and extending the useful lifetime of purified preparations .

How does the P. syringae RecTE-ligB system compare to other bacterial recombineering systems?

The P. syringae RecTE-ligB recombineering system exhibits distinct characteristics when compared to other bacterial genetic engineering platforms:

The P. syringae RecTE-ligB system offers several advantages for Pseudomonas engineering:

• Native compatibility with Pseudomonas cellular machinery, avoiding the need for extensive adaptation required for λ Red systems in pseudomonads .

• RecT alone is sufficient for ssDNA recombination, while both RecE and RecT are required for efficient dsDNA recombination, providing flexibility in application design .

• The incorporation of the native ligB likely enhances recombination efficiency by optimally sealing nicks in the final recombination products.

• The system can be applied to diverse P. syringae strains without requiring strain-specific modifications, unlike some heterologous systems that need optimization for each bacterial species .

What are the advantages of using native P. syringae proteins over heterologous recombineering systems?

Using native P. syringae proteins for recombineering offers several significant advantages over heterologous systems:

• Evolutionary adaptation: Native RecE, RecT, and ligB proteins have co-evolved within the P. syringae cellular environment, resulting in optimized interactions with endogenous DNA replication and repair machinery. This evolutionary adaptation ensures more efficient recombination processes compared to heterologous proteins that may face compatibility issues .

• Simplified implementation: Unlike λ Red recombineering in P. aeruginosa, which requires very long primers or multi-fragment splicing-by-overlap-extension (SOE)-PCR to generate mutant alleles and initial transformation steps to introduce recombination genes, native systems can be applied more directly .

• Broader strain applicability: The native P. syringae recombineering proteins function effectively across various P. syringae strains without requiring strain-specific optimizations. Once a suicide vector is constructed, it can be used directly on many genetic backgrounds of P. syringae, enabling parallel mutation introduction into multiple strains or sequential creation of multiple mutations in a single strain .

• Reduced cellular stress: Heterologous protein expression can trigger stress responses in bacteria, potentially reducing recombination efficiency. Native proteins are less likely to activate these stress pathways, resulting in healthier cells during the recombineering process.

• Optimized protein expression: Native proteins typically exhibit proper folding, stability, and subcellular localization in their source organism, while heterologous proteins may require extensive codon optimization or expression condition adjustments.

• Temperature compatibility: Native P. syringae proteins function optimally at the lower temperatures preferred by Pseudomonas species (25-30°C), unlike E. coli-derived systems that may have evolved for optimal activity at higher temperatures (37°C).

• Enhanced coordination with DNA ligase: The native ligB enzyme likely exhibits optimal coordination with RecE/RecT homologs, potentially increasing the efficiency and fidelity of the final recombination product through proper nick sealing .

These advantages collectively contribute to higher recombination frequencies, improved precision, and more seamless integration of the native RecTE-ligB system into P. syringae genetic engineering workflows.

How might ligB be integrated with CRISPR-Cas systems for enhanced genome editing in Pseudomonas?

Integrating ligB with CRISPR-Cas systems presents a promising strategy for enhanced Pseudomonas genome editing by combining the precision of CRISPR targeting with optimized DNA repair. This integration can be achieved through several strategic approaches:

• Enhanced homology-directed repair (HDR):

  • Couple Cas9-induced double-strand breaks with a RecE/RecT/ligB repair system

  • Design the system to express ligB during the repair phase to efficiently seal nicks in repaired DNA

  • Optimize the timing of ligB expression to coincide with the completion of RecT-mediated strand annealing

  • This combined approach could significantly increase HDR efficiency over standard CRISPR-HDR methods

• Single-strand template repair enhancement:

  • For single nucleotide modifications, combine Cas9 nickase (creating single-strand breaks) with RecT-mediated ssDNA incorporation

  • Express ligB to efficiently seal the remaining nicks after RecT-mediated strand exchange

  • This approach reduces cellular toxicity associated with double-strand breaks while maintaining high editing precision

• All-in-one vector systems:

  • Design plasmids containing:

    • Inducible Cas9/Cas12a expression cassette

    • Guide RNA expression under control of a constitutive promoter

    • RecT, RecE, and ligB under the control of separately inducible promoters

    • Homology repair template

  • Sequential induction allows precise temporal control of cutting and repair processes

• Base editing enhancement:

  • Combine CRISPR base editors (fusion of Cas9 nickase with deaminase) with ligB

  • LigB enhances the repair efficiency after base modification and nicking

  • This approach could reduce indel formation often observed with base editors

• Prime editing adaptation:

  • Develop a Pseudomonas-optimized prime editing system utilizing pegRNA principles

  • Incorporate ligB to efficiently seal nicks created during the prime editing process

  • The native ligB would likely perform better than the endogenous ligases in completing the prime editing reaction

This integration strategy leverages the targeting precision of CRISPR-Cas systems with the highly efficient native DNA repair machinery including ligB, potentially overcoming current limitations in Pseudomonas genome editing efficiency and precision . Experimental validation could focus on comparing editing efficiencies of standard CRISPR methods versus CRISPR-RecTE-ligB combined approaches across various Pseudomonas strains and editing contexts.

What emerging technologies might expand applications of ligB in synthetic biology?

Several emerging technologies are poised to expand the applications of ligB in synthetic biology, creating new opportunities for genetic manipulation and biological circuit design:

• Cell-free expression systems:

  • Developing Pseudomonas-derived cell-free protein synthesis platforms incorporating ligB

  • Creating rapid prototyping systems for testing recombination circuits before cellular implementation

  • Enabling directed evolution of ligB in cell-free environments for enhanced performance

  • Applications in biosensing and biomanufacturing outside living cells

• DNA nanotechnology integration:

  • Utilizing ligB for the assembly of complex DNA nanostructures optimized for bacterial systems

  • Creating ligB variants with altered sequence specificity for programmable DNA circuit assembly

  • Developing self-assembling DNA scaffolds that recruit ligB and other recombination proteins

  • Enabling spatial organization of synthetic metabolic pathways through DNA-scaffold engineering

• Microfluidic-based directed evolution:

  • High-throughput droplet systems for evolving ligB variants with novel properties

  • Coupling with fluorescence-activated droplet sorting for rapid selection

  • Developing continuous evolution platforms specific for DNA ligases

  • Creating specialized ligB variants for targeted biotechnology applications

• In vivo biosensors and circuit components:

  • Engineering ligB-based protein switches responsive to small molecules

  • Creating split-ligB systems for protein-protein interaction detection

  • Developing ligase-based logic gates for complex cellular computation

  • Incorporating ligB into synthetic genetic oscillators and timers

• Minimal genome initiatives:

  • Incorporating optimized ligB as a core component in minimal Pseudomonas genomes

  • Engineering streamlined DNA repair systems based on RecE/RecT/ligB for synthetic cells

  • Developing orthogonal DNA replication and repair systems using evolved ligB variants

  • Creating chassis organisms with enhanced genome stability for biotechnology applications

• Single-cell recombineering:

  • Developing techniques for targeted genetic modifications in individual bacteria

  • Combining with microfluidics and optical trapping for precise manipulation

  • Creating cellular barcoding systems using ligB-mediated recombination

  • Enabling evolutionary lineage tracking through progressive genomic modifications

These emerging technologies represent promising frontiers for expanding ligB applications beyond traditional recombineering, potentially revolutionizing our ability to engineer Pseudomonas and other bacteria for scientific research and biotechnological applications .

What unanswered questions remain about ligB structure-function relationships?

Despite progress in understanding recombinant ligB from P. syringae pv. tomato, several crucial questions about its structure-function relationships remain unanswered:

• Domain dynamics during catalysis:

  • How do the N-terminal, central, and C-terminal domains of ligB coordinate movements during the ligation reaction?

  • What conformational changes occur between the adenylation and nick-sealing steps?

  • Are there metastable intermediates that have not been captured in structural studies?

  • How does DNA binding trigger rearrangements in the active site architecture?

• Cofactor specificity determinants:

  • Which specific residues dictate the strict preference for NAD+ over ATP?

  • Are there allosteric sites that modulate cofactor binding or catalysis?

  • How does the protein distinguish between various NAD+ analogs?

  • Could engineered variants accept alternative cofactors for biotechnology applications?

• DNA substrate recognition mechanisms:

  • What structural features enable ligB to recognize nicked DNA?

  • How does the enzyme distinguish between various DNA structures (nicks, gaps, mismatches)?

  • Which protein-DNA interactions are essential versus dispensable for catalysis?

  • Are there sequence preferences that influence binding or catalytic efficiency?

• Interface with recombination machinery:

  • How does ligB physically interact with RecE and RecT proteins during recombination?

  • Are there specific protein-protein interactions that coordinate these activities?

  • Does RecT binding to DNA create preferred substrates for ligB?

  • How is ligB recruitment to recombination sites regulated?

• Regulatory mechanisms:

  • Are there post-translational modifications that regulate ligB activity in vivo?

  • How is ligB expression controlled during various cellular stress responses?

  • Does ligB participate in protein complexes that modulate its activity?

  • Are there bacterial regulatory proteins that directly interact with ligB?

• Evolution and specialization:

  • How has ligB evolved specifically in Pseudomonas compared to other bacterial ligases?

  • Which residues represent adaptations to the Pseudomonas cellular environment?

  • What selective pressures have shaped ligB's catalytic properties?

  • How does ligB compare with the primary DNA ligase (ligA) in terms of specialized functions?

Addressing these questions would not only advance fundamental understanding of DNA ligase biology but also inform rational engineering efforts to enhance ligB utility in recombineering and other biotechnological applications .

How might high-throughput approaches accelerate ligB engineering for biotechnology applications?

High-throughput approaches can dramatically accelerate ligB engineering for biotechnology applications by enabling rapid exploration of sequence-function relationships and identification of optimized variants:

• Deep mutational scanning:

  • Generate comprehensive single-mutation libraries covering the entire ligB sequence

  • Develop selection systems that link ligB activity to cell survival or fluorescent readouts

  • Utilize next-generation sequencing to identify activity-enhancing mutations

  • Create sequence-function maps to guide rational engineering efforts

  • This approach could identify mutations conferring enhanced thermostability, altered substrate specificity, or increased catalytic efficiency

• Microfluidic droplet-based screening:

  • Encapsulate individual bacteria expressing ligB variants in picoliter droplets

  • Include fluorescent DNA substrates that report on ligase activity

  • Sort droplets based on fluorescence intensity using droplet sorters

  • Achieve screening rates of >10⁶ variants per hour

  • This system would enable identification of rare high-activity variants from vast libraries

• Continuous directed evolution:

  • Develop phage-assisted continuous evolution (PACE) systems for ligB

  • Link successful DNA ligation to phage propagation through engineered genetic circuits

  • Perform hundreds of rounds of evolution in days rather than months

  • Apply selective pressures favoring desired properties (stability, specificity, etc.)

  • This approach could yield ligB variants with novel capabilities through exploration of fitness landscapes inaccessible to conventional methods

• Cell-free high-throughput expression:

  • Express ligB variant libraries in cell-free systems in 384 or 1536-well formats

  • Utilize automated liquid handling for rapid setup and analysis

  • Implement real-time activity monitoring using fluorescent or luminescent readouts

  • Bypass transformation bottlenecks of in vivo systems

  • This method could rapidly characterize thousands of variants under diverse conditions

• Machine learning-guided design:

  • Train algorithms on existing ligB sequence-function data

  • Generate in silico predictions of beneficial mutation combinations

  • Prioritize promising variants for experimental testing

  • Implement iterative design-build-test-learn cycles with decreasing library sizes and increasing precision

  • This approach could identify non-obvious mutation combinations that synergistically enhance desired properties

• Multiplex genome editing:

  • Apply CRISPR-based technologies for simultaneous testing of multiple ligB variants

  • Create barcoded strain libraries with different ligB mutations

  • Perform competitive growth assays under selective conditions

  • Use deep sequencing to track enrichment/depletion of variants

  • This strategy could identify ligB variants with optimal in vivo performance in relevant contexts

These high-throughput approaches would transform ligB engineering from a laborious, incremental process to a systematic exploration of protein function space, accelerating development of specialized variants for applications in recombineering, DNA nanotechnology, and synthetic biology .

What are recommended protocols for new researchers working with recombinant ligB?

For researchers new to working with recombinant ligB from P. syringae pv. tomato, the following detailed protocols are recommended:

Expression and Purification Protocol:

Materials:

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

• pET-based expression vector containing ligB with N-terminal His6-tag

• LB medium and appropriate antibiotics

• IPTG (isopropyl β-D-1-thiogalactopyranoside)

• Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 1 mM PMSF

• Wash buffer: Lysis buffer + 20 mM imidazole

• Elution buffer: Lysis buffer + 250 mM imidazole

• Dialysis buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol

• Ni-NTA resin

Procedure:

• Transform expression vector into BL21(DE3) using standard heat-shock protocol

• Grow transformed cells in LB medium with antibiotics at 37°C until OD600 reaches 0.6-0.8

• Cool culture to 18°C and induce with 0.5 mM IPTG

• Continue incubation at 18°C for 16-18 hours with shaking

• Harvest cells by centrifugation (5,000 × g, 15 min, 4°C)

• Resuspend pellet in lysis buffer (5 mL per gram of wet cell weight)

• Lyse cells by sonication (6 × 30 sec pulses with 30 sec cooling)

• Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

• Incubate supernatant with Ni-NTA resin (2 hours at 4°C with gentle agitation)

• Pack resin into column and wash with 10 column volumes of wash buffer

• Elute protein with 5 column volumes of elution buffer

• Dialyze against dialysis buffer overnight at 4°C

• Concentrate using centrifugal concentrators if necessary

• Verify purity by SDS-PAGE and measure concentration by Bradford assay

Basic LigB Activity Assay Protocol:

Materials:

• Purified recombinant ligB

• NAD+ (nicotinamide adenine dinucleotide)

• Linear nicked DNA substrate (e.g., pUC19 nicked with Nt.BbvCI)

• Reaction buffer: 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 50 μg/mL BSA

• Loading buffer with EDTA to stop reactions

• Agarose gel electrophoresis equipment

Procedure:

• Prepare 20 μL reaction mixtures containing:

  • 50 mM Tris-HCl pH 7.5

  • 10 mM MgCl2

  • 1 mM DTT

  • 50 μg/mL BSA

  • 1 mM NAD+

  • 100 ng nicked plasmid DNA

  • Various concentrations of purified ligB (10-500 ng)

• Incubate reactions at 30°C for 30 minutes

• Stop reactions by adding 5 μL of loading buffer containing 50 mM EDTA

• Analyze products by 0.8% agarose gel electrophoresis

• Stain with ethidium bromide and visualize under UV light

• Quantify the conversion of nicked to supercoiled DNA

Recombineering Protocol Using LigB with RecE/RecT:

Materials:

• P. syringae strain expressing RecE and RecT proteins

• Purified recombinant ligB (optional for exogenous addition)

• Linear DNA with 40-50 bp homology arms to target region

• Electroporation equipment and cuvettes

• Recovery medium (KB or similar)

• Selection plates with appropriate antibiotics

Procedure:

• Grow P. syringae cells expressing RecE/RecT to mid-log phase (OD600 0.4-0.6)

• Induce RecE/RecT expression if using an inducible system

• Prepare cells for electroporation:

  • Harvest 5 mL culture by centrifugation (4,000 × g, 5 min, 4°C)

  • Wash three times with ice-cold 10% glycerol

  • Resuspend in 100 μL 10% glycerol

• Mix 100 ng of linear DNA with competent cells

• For exogenous ligB addition, include 100-500 ng purified protein

• Transfer to pre-chilled electroporation cuvette (0.2 cm gap)

• Electroporate using standard Pseudomonas settings (e.g., 2.5 kV, 200 Ω, 25 μF)

• Immediately add 1 mL recovery medium

• Incubate at 28°C with shaking for 2-3 hours

• Plate various dilutions on selective media

• Incubate at 28°C for 2-3 days

• Screen resulting colonies by PCR and sequence verification

These protocols provide a foundation for new researchers to successfully work with recombinant ligB in various experimental contexts, from basic biochemical characterization to applied genetic engineering applications .

What interdisciplinary skills are most valuable for researchers working with ligB systems?

Researchers working with ligB systems benefit from developing a diverse set of interdisciplinary skills that span multiple scientific domains:

• Molecular Biology and Cloning:

  • Proficiency in PCR optimization for challenging GC-rich Pseudomonas templates

  • Expertise in designing efficient cloning strategies for large proteins

  • Ability to construct expression vectors with various tags and inducible promoters

  • Experience with site-directed mutagenesis techniques for structure-function studies

  • Skills in genomic DNA isolation from various Pseudomonas species

• Protein Biochemistry:

  • Expertise in protein expression optimization in bacterial systems

  • Proficiency in multi-step protein purification techniques

  • Experience with protein characterization methods (circular dichroism, thermal shift assays)

  • Skills in enzyme kinetics analysis and data interpretation

  • Ability to troubleshoot protein stability and solubility issues

• Structural Biology:

  • Understanding of protein crystallization principles and techniques

  • Ability to interpret X-ray crystallography or cryo-EM data

  • Experience with molecular modeling and structural prediction tools

  • Skills in analyzing protein-substrate interactions and dynamics

  • Knowledge of structural visualization software for hypothesis generation

• Microbiology:

  • Proficiency in culturing and maintaining Pseudomonas strains

  • Experience with bacterial transformation and electroporation optimization

  • Understanding of bacterial growth kinetics and physiological states

  • Knowledge of antibiotic selection strategies for various genetic backgrounds

  • Skills in phenotypic characterization of recombinant strains

• Bioinformatics:

  • Ability to perform sequence alignments and phylogenetic analyses of ligases

  • Experience with genomic data mining to identify novel ligB homologs

  • Proficiency in using structure prediction algorithms and molecular dynamics tools

  • Skills in designing primers for complex genetic manipulations

  • Understanding of next-generation sequencing data analysis for validation studies

• Data Analysis and Statistics:

  • Expertise in experimental design to ensure statistical validity

  • Proficiency in using statistical software for analyzing enzymatic assay data

  • Skills in developing reproducible data analysis workflows

  • Experience with visualization tools for complex datasets

  • Understanding of appropriate statistical tests for various experimental scenarios

• Synthetic Biology Principles:

  • Knowledge of genetic circuit design and optimization

  • Understanding of DNA assembly methods beyond traditional cloning

  • Experience with standardized biological parts and modular design

  • Skills in characterizing and troubleshooting genetic constructs

  • Ability to implement feedback control in biological systems

Researchers who develop this interdisciplinary skill set are better positioned to overcome the technical challenges associated with ligB systems and can more effectively apply these enzymes in novel research contexts, from fundamental biochemistry to cutting-edge synthetic biology applications .

How can research on ligB be integrated into advanced molecular biology curriculum?

Integrating ligB research into advanced molecular biology curriculum offers valuable opportunities for comprehensive training in modern biotechnology. Here's a structured approach for educational integration:

Course Module Development:

Laboratory Course: "Recombinant Protein Engineering and Applications"

• Week 1-2: Cloning ligB into expression vectors, introducing site-directed mutations

• Week 3-4: Expression and purification of wildtype and mutant ligB proteins

• Week 5-6: Activity assays and biochemical characterization

• Week 7-8: Application in recombineering experiments with RecE/RecT

• Week 9-10: Data analysis, presentation, and scientific writing

Lecture Series: "Enzyme Structure-Function in Genome Engineering"

• Comparative analysis of DNA ligases across domains of life

• Mechanistic details of phosphodiester bond formation

• Evolution of NAD+ versus ATP-dependent ligases

• Integration of ligases in DNA repair and recombination pathways

• Case studies of ligB applications in synthetic biology

Research-Based Learning Projects:

Multi-step Projects for Advanced Undergraduates:

• "Engineering ligB variants with altered cofactor preference"

  • Apply structure-guided mutagenesis to modify the NAD+-binding pocket

  • Characterize mutants biochemically

  • Test functionality in DNA repair assays

• "Optimizing recombineering efficiency in Pseudomonas using ligB"

  • Test various expression levels and conditions

  • Develop quantitative recombination frequency assays

  • Compare outcomes with and without ligB supplementation

• "Investigating ligB substrate preferences through synthetic DNA designs"

  • Create DNA substrates with systematic variations

  • Analyze ligation efficiency using gel-based assays

  • Correlate structural features with enzymatic activity

Interdisciplinary Integration:

Bioinformatics Component:

• Database mining for novel ligB homologs in bacterial genomes

• Structural prediction and comparative analysis

• Primer design for targeted mutagenesis

• Analysis of protein-protein interaction networks

Bioethics Discussion:

• Implications of engineered recombination systems

• Biosafety considerations for enhanced genetic modification tools

• Intellectual property issues in enzyme engineering

• Responsible innovation in genome editing technologies

Assessment Strategies:

Skill-Based Evaluation:

• Laboratory notebooks documenting experimental design and troubleshooting

• Technical proficiency in protein expression and enzymatic assays

• Data analysis and interpretation of complex biochemical results

• Scientific communication through research reports and presentations

Concept Integration Assessment:

• Case studies requiring application of ligB knowledge to novel problems

• Experimental design proposals addressing real research challenges

• Peer review of classmates' research reports using journal review criteria

• Synthesis of findings across multiple experiments into a cohesive narrative

Resources and Materials:

Laboratory Manual:

• Detailed protocols for ligB cloning, expression, and characterization

• Troubleshooting guides for common technical challenges

• Data analysis templates and examples

• Safety considerations specific to recombinant DNA work

Digital Learning Tools:

• Interactive structural models of ligB highlighting functional domains

• Simulation software for predicting effects of mutations

• Video demonstrations of advanced purification techniques

• Online forum for collaborative problem-solving