Recombinant Vibrio vulnificus Primosomal replication protein n (priB)

What is PriB and what is its role in DNA replication in Vibrio vulnificus?

PriB is a primosomal protein required for the replication fork restart in bacteria. In prokaryotes like Vibrio vulnificus, PriB plays a crucial role in the DNA replication process, particularly when replication forks encounter DNA damage or stalled replication. PriB is the second protein to participate in the protein-DNA complex formation in a PriA-PriB-DnaT-dependent reaction . This protein significantly contributes to genetic integrity maintenance after encountering DNA damage, which is essential for bacterial survival.

Methodologically, to establish PriB's role in V. vulnificus, researchers typically employ gene deletion studies combined with viability assessments under DNA-damaging conditions. These experiments should include controls such as complementation with recombinant PriB to confirm phenotypic rescue.

How does PriB's function differ from other DNA-binding proteins in Vibrio vulnificus?

While PriB shares structural similarity with single-stranded DNA-binding proteins (SSB), they bind ssDNA differently. The crystal structures reveal that ssDNA wraps around SSB in a binding topology resembling seams on a baseball, while ssDNA adopts an Ω-shaped conformation when binding to one monomer of the PriB dimer .

This functional difference can be demonstrated experimentally using electrophoretic mobility shift analysis (EMSA), which reveals different ssDNA-binding patterns between SSB and PriB. SSB forms multiple distinct complexes with ssDNA of different lengths, whereas PriB binding to ssDNA of different lengths only forms a single complex .

What are the key structural features of V. vulnificus PriB and how do they relate to its function?

V. vulnificus PriB presents as a homodimer with two oligonucleotide/oligosaccharide-binding (OB) folds . The most apparent structural difference between PriB and its evolutionary ancestor SSB is the absence of the C-terminal protein-protein interaction domain (SSBc) in PriB.

The structural organization of PriB directly influences its ssDNA binding capability and interaction with other proteins in the replication restart primosome. Research has shown that despite not possessing SSBc, PriB can still stimulate the activity of PriA , indicating unique structural adaptations that compensate for this absence.

To investigate these structural properties, researchers typically employ X-ray crystallography, circular dichroism spectroscopy, and analytical ultracentrifugation techniques.

How do PriB proteins from different Vibrio species compare structurally and functionally?

While the search results don't provide direct comparisons between PriB proteins from different Vibrio species, comparative genomic and structural analyses would be valuable. The approach to this question would involve:

• Heterologous expression of PriB from various Vibrio species (including V. vulnificus, V. parahaemolyticus, V. cholerae)

• Structural determination using X-ray crystallography

• Functional comparison using DNA binding assays and PriA stimulation assays

• Sequence alignment and phylogenetic analysis

This research would help understand species-specific adaptations of PriB and potential correlations with pathogenicity or environmental adaptation.

What are the most effective experimental designs for studying V. vulnificus PriB function in vitro?

For robust in vitro analysis of V. vulnificus PriB function, a multi-technique approach is recommended:

• DNA binding studies: EMSA, fluorescence anisotropy, and surface plasmon resonance to determine binding affinities and kinetics

• Protein-protein interaction studies: Pull-down assays, bacterial two-hybrid systems, and isothermal titration calorimetry to analyze interactions with PriA and other primosomal proteins

• Enzymatic assays: Helicase assays to measure PriB's effect on PriA helicase activity

• Structural studies: X-ray crystallography of PriB-DNA and PriB-protein complexes

An effective experimental design would include positive controls (such as E. coli PriB), negative controls (such as mutant PriB lacking DNA binding ability), and appropriate buffer conditions optimized for V. vulnificus PriB stability and activity.

How can we determine the oligomerization state of PriB in solution?

Based on research with other PriB proteins, analytical approaches to determine the oligomerization state include:

• Gel filtration chromatography: This technique separates proteins based on size and shape. A study of PriB-SSBc showed a single peak with an elution volume corresponding to a dimer rather than a tetramer .

• Analytical ultracentrifugation: This provides precise determination of molecular mass in solution without reference to standards.

• Native PAGE: This allows visualization of the native oligomeric state.

• Cross-linking experiments: Chemical cross-linking followed by SDS-PAGE can capture transient interactions and confirm stable oligomeric states.

For example, in a study of chimeric PriB-SSBc, gel filtration chromatography showed a single peak with an elution volume of 90.5 mL, corresponding to a molecular mass of 34,209 Da, which is approximately twice the calculated monomeric mass, confirming its dimeric nature .

What is the optimal expression system for producing recombinant V. vulnificus PriB?

For optimal expression of recombinant V. vulnificus PriB, the following system has proven effective:

• Expression vector: A pET system vector (such as pET21a) with an inducible T7 promoter is recommended, similar to successful expressions of other recombinant Vibrio proteins .

• Host strain: E. coli BL21(DE3) has been successfully used for expression of Vibrio proteins, as it lacks certain proteases and provides tight control of T7 promoter-driven expression .

• Induction conditions: Optimal conditions typically include growth at 37°C until OD600 reaches 0.5-0.6, followed by induction with 0.5 mM IPTG, with subsequent growth at a lower temperature (16-25°C) to enhance proper folding .

• Affinity tags: An N-terminal or C-terminal His-tag (6× or 10×) facilitates purification while minimally affecting protein function .

What purification strategy yields the highest purity and activity of recombinant V. vulnificus PriB?

A multi-step purification approach is recommended:

• Initial clarification: After cell lysis, centrifugation at 15,000-20,000 × g for 30 minutes to remove cell debris.

• Affinity chromatography: Using Ni-Sepharose columns for His-tagged PriB, with careful optimization of imidazole concentrations in binding and elution buffers .

• Ion exchange chromatography: SP or Q column chromatography depending on the isoelectric point of V. vulnificus PriB. For example, KpPriB was successfully purified using an SP column by the AKTA-FPLC system .

• Size exclusion chromatography: A final polishing step using Superdex 75 or 200 can remove aggregates and yield highly pure protein.

• Buffer optimization: Based on similar proteins, a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, and 5% glycerol is likely suitable for maintaining stability and activity.

Typical yields from this approach should be 10-20 mg of purified protein per liter of culture, with purity exceeding 95% as assessed by SDS-PAGE.

What methodologies provide the most accurate measurement of PriB-DNA binding affinities?

Multiple complementary approaches are recommended for accurate measurement of PriB-DNA binding affinities:

• Electrophoretic Mobility Shift Assay (EMSA): This technique can provide apparent Kd values and reveal binding patterns. For PriB-ssDNA interactions, using radiolabeled or fluorescently labeled oligonucleotides of varying lengths (20-70 nucleotides) is recommended .

• Fluorescence Anisotropy: This provides real-time binding data and is particularly useful for determining binding kinetics. Use fluorescein-labeled ssDNA with increasing concentrations of PriB (0.1 nM to 1 μM).

• Isothermal Titration Calorimetry (ITC): This gives a complete thermodynamic profile of binding, including Kd, stoichiometry, enthalpy, and entropy changes.

• Surface Plasmon Resonance (SPR): This allows determination of association and dissociation rate constants (kon and koff).

When conducting these experiments, it's critical to control buffer conditions (salt concentration significantly affects DNA binding) and to include positive controls (e.g., established DNA-binding proteins) and negative controls (e.g., non-DNA binding proteins).

How does DNA sequence and structure affect PriB binding?

While specific data for V. vulnificus PriB is not provided in the search results, research with other PriB proteins suggests:

• Sequence preference investigation: Test binding to random sequence oligonucleotides versus designed sequences with varying GC content.

• Structure preference analysis: Compare binding to ssDNA, dsDNA, and DNA structures with forks, bubbles, and overhangs.

• Competition assays: Use unlabeled DNA of various sequences and structures to compete with labeled standard DNA.

• Footprinting: Deploy DNase I footprinting or hydroxyl radical footprinting to identify specific contacts between PriB and DNA.

The PriB-DNA crystal structure would be particularly informative, revealing the binding interface and specific amino acid-nucleotide interactions.

How can we investigate PriB's interaction with other replication restart proteins in V. vulnificus?

To study PriB's interactions with other replication restart proteins, particularly PriA:

• Pull-down assays: Using His-tagged PriB to pull down interacting partners from V. vulnificus cell lysates, followed by mass spectrometry identification.

• Bacterial two-hybrid system: To detect direct protein-protein interactions in vivo.

• Surface Plasmon Resonance (SPR): To determine binding kinetics and affinities between purified PriB and potential partners.

• Protein crosslinking: Using chemical crosslinkers followed by mass spectrometry to identify interaction interfaces.

• Functional assays: For example, testing how PriB affects PriA helicase activity using radiolabeled DNA substrates and thin-layer chromatography to detect ATP hydrolysis.

Research with related proteins has shown that PriB can induce conformational changes in PriA, significantly stimulate PriA activity, and facilitate the association of DnaT with PriA .

What is the mechanism by which PriB stimulates PriA activity in replication restart?

While the exact mechanism for V. vulnificus PriB is not detailed in the search results, based on related systems:

• Conformational change hypothesis testing: Use limited proteolysis, circular dichroism, or FRET to detect PriB-induced conformational changes in PriA.

• DNA substrate modification analysis: Investigate whether PriB alters the structure of DNA substrates to make them more accessible to PriA.

• Order-of-addition experiments: Determine whether PriB must bind DNA before PriA, or vice versa, for optimal stimulation.

• Domain mapping: Create truncation or point mutants of both PriB and PriA to identify domains critical for their interaction and functional stimulation.

Similar studies have shown that PriB can induce conformational alterations in PriA, significantly stimulate the activity of PriA, and facilitate the association of DnaT with PriA .

How can we design PriB mutants to investigate structure-function relationships?

Design of informative PriB mutants requires careful consideration of structural and evolutionary data:

• Conserved residue targeting: Identify residues conserved across PriB proteins but distinct from SSB proteins. This may point to PriB-specific functions.

• Interface disruption: Based on structural data, mutate residues involved in:

  • Dimerization interface

  • DNA binding interface

  • Predicted protein-protein interaction sites

• Domain swapping: Create chimeric proteins by swapping domains between PriB and SSB to investigate domain-specific functions, similar to the PriB-SSBc chimeric study .

• Site-directed mutagenesis approach: Use overlap extension PCR or commercial site-directed mutagenesis kits to introduce specific mutations.

• Validation strategy: Confirm structural integrity of mutants using circular dichroism, thermal shift assays, and size exclusion chromatography before functional testing.

A successful example is the creation of the chimeric PriB-SSBc protein, which demonstrated that SSBc could significantly enhance the ssDNA-binding affinity, change the binding behavior, and further stimulate the PriA activity of PriB .

What approaches can distinguish between the direct and indirect effects of PriB on replication restart?

Distinguishing direct from indirect effects requires sophisticated experimental designs:

• Purified component reconstitution: Reconstitute the minimal replication restart system with purified components to observe direct effects of PriB.

• Order-of-addition experiments: Systematically vary the order of addition of components to identify dependencies.

• Temporal resolution techniques: Use rapid-mixing techniques (stopped-flow, quench-flow) to identify the kinetically relevant steps affected by PriB.

• Single-molecule approaches: Apply FRET or optical tweezers to directly observe individual molecular events.

• In vivo reporter systems: Develop fluorescent reporters that respond to specific steps in replication restart to monitor the process in living cells.

How do we address disparities in reported PriB-DNA binding affinities across different studies?

Disparities in DNA binding affinities are common and require careful analysis:

• Standardization of experimental conditions: Differences in buffer composition (particularly salt concentration), temperature, pH, and protein/DNA concentrations can dramatically affect binding measurements.

• Method-specific biases: Different techniques (EMSA, fluorescence anisotropy, ITC, SPR) have inherent biases. For example, EMSA may underestimate Kd for fast-dissociating complexes.

• Reconciliation approach:

  • Replicate studies using multiple techniques

  • Directly compare different PriB proteins under identical conditions

  • Use mathematical models to normalize data across different experimental conditions

• Reporting standards: Include detailed methods sections specifying all buffer components, protein preparation methods, and data analysis approaches.

Consider that true biological variability may exist; for example, the ssDNA-binding affinity of PriB is significantly lower (>2–3 orders of magnitude) than that of SSB proteins , which may reflect their different biological roles.

What explains the variation in PriB's ability to stimulate PriA between different experimental systems?

Variation in PriB stimulation of PriA could result from several factors:

• Protein source variability: Differences in expression systems, purification methods, and storage conditions can affect protein activity.

• DNA substrate differences: The sequence, structure, and length of DNA substrates can dramatically influence helicase activity and stimulation.

• Assay condition differences: ATP concentration, divalent cation type and concentration, temperature, and salt concentration all affect helicase activity.

• Experimental approach:

  • Use common DNA substrates across studies

  • Include internal controls for activity normalization

  • Directly compare proteins under identical conditions

  • Consider the influence of additional factors present in some experimental systems but not others

• Domain-specific effects: As demonstrated by the PriB-SSBc chimeric study, specific domains can significantly influence functional interactions .

How might studying PriB contribute to developing antimicrobial strategies against V. vulnificus?

V. vulnificus is a significant pathogen with high fatality rates that has shown increasing antibiotic resistance . Targeting PriB offers several potential therapeutic advantages:

• Essentiality assessment: Determine whether PriB is essential for V. vulnificus virulence through gene deletion studies in infection models. Replication restart becomes particularly important under stress conditions encountered during infection.

• High-throughput screening: Develop assays suitable for screening chemical libraries for inhibitors of:

  • PriB-DNA interaction

  • PriB-PriA interaction

  • PriB-stimulated PriA activity

• Structure-based drug design: Use the crystal structure of V. vulnificus PriB to identify binding pockets suitable for small molecule targeting.

• Selectivity consideration: Compare the structures of bacterial PriB with human ssDNA-binding proteins to identify differences that could be exploited for selective targeting.

• Combination therapy potential: Investigate whether PriB inhibitors might synergize with conventional antibiotics, particularly under DNA-damaging conditions.

Recent research has shown that antibiotic resistance patterns among V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009 , suggesting that novel targets like PriB could be valuable for addressing future resistance challenges.

What novel experimental techniques could advance our understanding of PriB function in V. vulnificus?

Cutting-edge approaches to advance PriB research include:

• Cryo-electron microscopy: For visualizing larger complexes involving PriB, particularly those that might be difficult to crystallize.

• Single-molecule techniques: Including FRET, optical tweezers, and magnetic tweezers to observe individual molecular events during replication restart.

• Live-cell imaging: Using fluorescently tagged PriB to track its localization and dynamics in living V. vulnificus cells, particularly under DNA-damaging conditions.

• ChIP-seq: To identify genomic binding sites of PriB in vivo, particularly at stalled replication forks.

• CRISPR interference (CRISPRi): For tunable repression of PriB in vivo to assess dose-dependent phenotypes.

• Proximity labeling: Using technologies like BioID or APEX to identify proteins that interact with PriB in vivo, potentially revealing previously unknown partners.

• Microfluidic approaches: For high-throughput assessment of PriB function under various stress conditions.