Recombinant Bacillus subtilis Putative DNA relaxase nicK (nicK)

What is DNA relaxase nicK and what is its primary function in Bacillus subtilis?

DNA relaxase nicK belongs to a class of enzymes essential for bacterial conjugative DNA transfer. In Bacillus subtilis, relaxases play a crucial role in initiating and terminating conjugative DNA transfer by catalyzing site-specific nicking reactions . These proteins function through a transesterification mechanism, creating a nick in duplex DNA in a site- and strand-specific manner. The reaction results in a nicked double-stranded DNA molecule with a sequestered 3'-OH end and the relaxase covalently attached to the 5' end of the cleaved strand through a phosphotyrosyl linkage . This covalent nucleoprotein complex serves as an intermediate in conjugative DNA transfer, allowing for the subsequent unwinding and transfer of a single DNA strand to recipient cells.

How does the catalytic mechanism of DNA relaxase nicK differ from other DNA modifying enzymes?

The catalytic mechanism of DNA relaxase nicK is distinctive for several key reasons:

• The relaxase catalyzes an isoenergetic and reversible transesterification reaction, unlike many restriction enzymes that perform hydrolytic cleavage .

• The enzyme forms a covalent intermediate with DNA through a phosphotyrosyl bond, creating a relatively long-lived nucleoprotein complex essential for DNA transfer .

• The relaxase can perform both DNA nicking (first transesterification) and ligation (second transesterification), making it functionally different from nucleases that only cleave DNA .

These characteristics make relaxases uniquely suited for their role in conjugative transfer, as they initiate strand transfer through the first transesterification and terminate the process through the second .

What recognition sequences are targeted by DNA relaxase nicK in Bacillus subtilis?

Research indicates that DNA relaxase nicK recognition sites in Bacillus subtilis typically occur in regions with relatively high AT content . Studies on illegitimate plasmid recombination in B. subtilis have suggested that DNA topoisomerase I-dependent single-strand DNA nicking may be the primary source of type I deletions between non-repeated DNA sequences . Analysis of recombination frequencies using positive selection vectors has helped identify potential recognition sites, which differ from the homologous recombination sites requiring extensive sequence similarity . The specificity of these recognition sites is critical for the proper function of relaxases in conjugative DNA transfer systems.

Which expression systems yield optimal results for producing recombinant DNA relaxase nicK?

According to research data, multiple expression systems can be used for producing recombinant putative DNA relaxase nicK, with E. coli and yeast systems offering the best yields and shorter turnaround times . The choice of expression system should be guided by specific research requirements:

For most biochemical characterization studies, E. coli expression systems provide sufficient yields of functional protein, while maintaining activity and structural integrity suitable for research purposes .

What purification strategy provides the highest yield of active recombinant DNA relaxase nicK?

Based on published protocols, an effective purification strategy for recombinant DNA relaxase nicK involves:

• Affinity chromatography using nickel columns for His-tagged constructs, which yields >95% pure protein .

• Collection of E. coli cells after IPTG induction (typically 1 mM IPTG for 2 hours at OD₆₀₀ of 0.6) .

• Cell lysis followed by clarification of lysate through centrifugation .

• Nickel-column purification of the His-tagged protein .

• Dialysis against an appropriate buffer (e.g., 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 500 mM NaCl, 10 mM MgCl₂, 7 mM β-mercaptoethanol, 50% v/v glycerol) .

• Storage of purified protein in aliquots at -80°C to maintain activity .

Protein concentration can be reliably determined using Bradford assay and OD₂₈₀ measurements .

How can researchers overcome expression challenges when producing recombinant DNA relaxase nicK?

When encountering expression challenges with recombinant DNA relaxase nicK, researchers should consider these methodological approaches:

• Optimize codon usage for the host expression system, particularly when expressing Bacillus subtilis proteins in E. coli.

• Adjust growth temperature during induction (often lower temperatures of 16-25°C improve soluble protein yield).

• Test various induction conditions by varying IPTG concentration (0.1-1 mM) and induction duration (2-16 hours) .

• Consider fusion tags that enhance solubility (such as MBP or SUMO) in addition to purification tags.

• Supplement growth media with cofactors that might stabilize the protein during expression.

• Explore co-expression with chaperones if misfolding is suspected.

These strategies can significantly improve the yield and activity of recombinant DNA relaxase nicK, particularly when dealing with challenging constructs or expression conditions.

What analytical methods are most effective for characterizing DNA relaxase nicK interactions with target DNA?

Several complementary analytical techniques provide valuable insights into relaxase-DNA interactions:

• Analytical Ultracentrifugation: Sedimentation velocity (SV) and sedimentation equilibrium (SE) experiments can characterize the hydrodynamic properties of relaxase-DNA complexes and provide information about complex formation and stoichiometry .

• Dynamic Light Scattering (DLS): This technique helps determine the size distribution of relaxase proteins and their complexes with DNA, providing insights into aggregation states and conformational changes upon DNA binding .

• Electrophoretic Mobility Shift Assays (EMSA): These assays visualize relaxase-DNA complexes based on their altered mobility in non-denaturing gels compared to free DNA.

• DNase I Footprinting: This method identifies specific DNA sequences protected by bound relaxase proteins.

• Surface Plasmon Resonance (SPR): SPR provides real-time binding kinetics and affinity measurements between relaxase and its DNA substrates.

These techniques, used in combination, provide comprehensive characterization of relaxase-DNA interactions, essential for understanding the mechanism of action .

How can researchers functionally assess the nicking activity of DNA relaxase nicK?

To accurately assess the nicking activity of DNA relaxase nicK, researchers should employ these methodological approaches:

• Supercoiled DNA relaxation assays: Monitor the conversion of supercoiled plasmid DNA to nicked open circular forms using agarose gel electrophoresis .

• Site-specific nicking assays: Use oligonucleotide substrates containing the putative recognition sequence to assess sequence specificity of nicking.

• Kinetic analysis: Measure the rate of nicking reaction under various conditions (pH, temperature, salt concentration) to determine optimal reaction parameters.

• Covalent protein-DNA complex detection: Use SDS-PAGE followed by either phosphorimaging (with labeled DNA) or Western blotting (with antibodies against the relaxase) to detect the covalent intermediate .

• Reversibility testing: Examine the ability of the relaxase to perform the second transesterification reaction (ligation) under various conditions .

These assays collectively provide a comprehensive assessment of relaxase activity, specificity, and mechanism.

What experimental systems best demonstrate the in vivo function of DNA relaxase nicK in Bacillus subtilis?

To effectively study in vivo function of DNA relaxase nicK in Bacillus subtilis, researchers should consider these experimental systems:

• Conjugation assays in liquid medium: These directly measure the transfer frequency of conjugative elements and can assess the impact of relaxase mutations or overexpression .

• Inducible expression systems: Using promoters like P₍ₓᵧₗ₎ or P₍ₛₚₐₙₖ₎ allows controlled expression of wild-type or mutant relaxase to study dosage effects on conjugation .

• Complementation studies: Expressing relaxase variants in relaxase-deficient strains to restore conjugation can identify essential functional domains.

• Fluorescently tagged relaxase tracking: Visualizing the subcellular localization of fluorescently labeled relaxase during conjugation provides insights into dynamics and interactions.

• Chromatin immunoprecipitation (ChIP): This technique identifies in vivo DNA binding sites of the relaxase within the bacterial genome.

These systems allow researchers to connect biochemical properties with biological function in the native bacterial context .

How is recombinant Bacillus subtilis DNA relaxase nicK being utilized in vaccine development research?

Research has demonstrated the potential of recombinant Bacillus subtilis expressing heterologous antigens as vaccine delivery vehicles. In this context, DNA relaxase nicK knowledge is applicable in several ways:

• Recombinant B. subtilis has been successfully engineered to express dominant antigen regions of viral proteins, such as pseudorabies virus (PRV) gC and gD proteins .

• Intranasal administration of such recombinant B. subtilis strains effectively stimulates both mucosal IgA and systemic IgG immune responses in animal models .

• The recombinant B. subtilis approach has shown promise in regulating specific T lymphocyte proliferative responses through cytokines like IFN-γ and IL-10 .

• This approach has proven effective in producing high titers of neutralizing antibodies against viral infections like PRV .

Understanding the mechanisms by which DNA relaxase nicK facilitates genetic engineering of B. subtilis enhances our ability to develop these bacterial vectors for vaccine delivery. This knowledge is particularly valuable for developing mucosal vaccines that can trigger both local and systemic immunity .

How does the study of DNA relaxase nicK contribute to understanding horizontal gene transfer in bacterial communities?

DNA relaxase nicK plays a fundamental role in conjugative DNA transfer, one of the primary mechanisms of horizontal gene transfer (HGT) in bacterial communities. Research in this area reveals:

• Relaxases initiate and terminate conjugative DNA transfer by catalyzing site- and strand-specific DNA nicking through transesterification reactions .

• The relaxase, together with auxiliary proteins, forms a relaxosome complex that processes DNA for transfer .

• Conjugative elements containing relaxase nicK can transfer between bacteria, contributing to genetic diversity and adaptation in bacterial communities.

• Understanding relaxase mechanisms provides insights into the spread of antibiotic resistance genes and virulence factors through bacterial populations.

• The study of relaxase nicK in B. subtilis offers perspectives on HGT in Gram-positive bacteria, which is less understood compared to Gram-negative systems .

This research contributes to our fundamental understanding of bacterial evolution and adaptation, with implications for controlling the spread of antibiotic resistance and developing strategies to manipulate bacterial communities in various environments.

What are the potential applications of recombinant DNA relaxase nicK in synthetic biology and genetic engineering?

Recombinant DNA relaxase nicK offers several promising applications in synthetic biology and genetic engineering:

• Development of novel site-specific DNA manipulation tools: The precise nicking activity of relaxases could be harnessed for targeted genetic modifications.

• Creation of improved bacterial vectors: Understanding relaxase function can lead to the development of more efficient plasmid vectors for gene delivery in various bacterial hosts.

• Engineering controlled horizontal gene transfer systems: Manipulated relaxase systems could allow for directed gene transfer between specific bacterial populations.

• Design of bacterial biosensors: Relaxase-based systems might be engineered to trigger DNA transfer in response to specific environmental signals.

• Development of probiotic strains with enhanced properties: Engineering B. subtilis with modified relaxase systems could create probiotic strains with improved colonization or therapeutic properties .

• Vaccine delivery platforms: As demonstrated with pseudorabies antigens, relaxase knowledge enhances our ability to develop B. subtilis as a vehicle for antigen delivery .

These applications leverage the natural DNA processing capabilities of relaxases to create new tools and systems for biotechnology and biomedicine.

How can researchers distinguish between DNA relaxase nicK activity and other nuclease contamination in experimental preparations?

Distinguishing relaxase activity from other nucleases requires careful experimental design:

• Substrate specificity testing: Relaxases typically nick at specific recognition sequences, while most contaminating nucleases show limited sequence specificity. Using substrates with and without the recognition sequence can help identify specific relaxase activity .

• Reversibility assessment: Unlike most nucleases, relaxases catalyze reversible reactions. Demonstrating DNA ligation under appropriate conditions strongly supports relaxase activity .

• Covalent intermediate detection: The formation of a covalent protein-DNA complex is characteristic of relaxases. Detecting this intermediate (e.g., by SDS-PAGE) confirms relaxase activity .

• Metal ion dependency: Characterizing the metal ion requirements can help distinguish relaxase activity from other nucleases, as they often have different cofactor preferences.

• Inhibitor profiling: Different classes of nucleases show distinct inhibition patterns with various inhibitors.

• Site-directed mutagenesis: Mutating the catalytic tyrosine of the relaxase should abolish activity without affecting contaminating nucleases.

These approaches collectively provide strong evidence for authentic relaxase activity.

What strategies help maintain enzymatic activity of recombinant DNA relaxase nicK during purification and storage?

To preserve the enzymatic activity of recombinant DNA relaxase nicK, researchers should consider:

• Buffer optimization: Include key components in purification and storage buffers:

  • 20 mM Tris-HCl pH 8.0 (maintains optimal pH)

  • 1 mM EDTA (chelates metal ions that could promote oxidation)

  • 500 mM NaCl (provides ionic strength and stability)

  • 10 mM MgCl₂ (cofactor for enzymatic activity)

  • 7 mM β-mercaptoethanol (prevents oxidation of cysteine residues)

  • 50% v/v glycerol (prevents freezing damage during storage)

• Temperature management: Maintain protein at 4°C during purification and store at -80°C for long-term preservation .

• Aliquoting: Divide purified protein into small aliquots to avoid repeated freeze-thaw cycles .

• Protein concentration: Maintain protein at moderate concentrations (1-5 mg/ml) to prevent aggregation while ensuring stability.

• Protease inhibitors: Include protease inhibitors during cell lysis and initial purification steps to prevent degradation.

• Quick processing: Minimize the time between cell lysis and final purification to limit exposure to cellular proteases and oxidizing conditions.

These strategies significantly enhance the stability and activity of purified recombinant DNA relaxase nicK.

How can researchers overcome challenges in studying relaxase-auxiliary protein interactions in the relaxosome complex?

Studying the interactions between relaxase nicK and auxiliary proteins in the relaxosome complex presents several challenges. These methodological approaches can help overcome them:

• Co-expression and co-purification: Express relaxase together with auxiliary proteins to capture native interactions . This approach has been successful with relaxase and auxiliary proteins from conjugative plasmid pLS20 .

• Analytical ultracentrifugation: Use sedimentation velocity (SV) and sedimentation equilibrium (SE) experiments to characterize complex formation and determine stoichiometry .

• Dynamic light scattering (DLS): Employ DLS to assess size distribution changes upon complex formation .

• Pull-down assays with tagged proteins: Use differentially tagged proteins (e.g., His-tag, GST-tag) to selectively pull down complexes and identify interacting partners.

• Crosslinking coupled with mass spectrometry: Apply chemical crosslinking followed by mass spectrometry to identify interaction interfaces between relaxase and auxiliary proteins.

• Bacterial two-hybrid systems: Utilize two-hybrid approaches adapted for bacterial proteins to screen for interactions in vivo.

• Fluorescence resonance energy transfer (FRET): Apply FRET to study dynamics of protein-protein interactions in real-time, potentially in living cells.

These approaches collectively provide a comprehensive picture of the relaxosome complex assembly and function .

What emerging technologies might enhance our understanding of DNA relaxase nicK structure-function relationships?

Several cutting-edge technologies show promise for advancing our understanding of DNA relaxase nicK:

• Cryo-electron microscopy (Cryo-EM): This rapidly advancing technique can reveal the structure of relaxase-DNA complexes in near-native conditions, potentially capturing different conformational states during the catalytic cycle.

• Single-molecule techniques: Methods such as optical tweezers, magnetic tweezers, or FRET can track relaxase activity at the single-molecule level, providing insights into reaction mechanisms and kinetics not observable in bulk assays.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach can map conformational changes and protein-DNA interaction surfaces with high resolution.

• AlphaFold and other AI-based structure prediction: These computational tools could predict structures of relaxase variants and complexes, guiding experimental design.

• CRISPR-based genome editing in B. subtilis: Advanced CRISPR techniques allow precise modification of relaxase genes in their native context to study function.

• Time-resolved X-ray crystallography: This emerging technique could capture transient intermediates during the relaxase reaction.

These technologies promise to bridge current knowledge gaps regarding the structural dynamics and catalytic mechanism of DNA relaxase nicK.

How might research on DNA relaxase nicK contribute to addressing antibiotic resistance challenges?

Research on DNA relaxase nicK has significant implications for addressing antibiotic resistance:

• Targeting conjugative transfer: Since relaxases are essential for conjugative transfer of antibiotic resistance genes, they represent potential targets for inhibitors that could limit the spread of resistance .

• Understanding resistance gene mobility: Research on relaxase mechanisms provides insights into how resistance genes move between bacteria, informing surveillance and containment strategies.

• Developing anti-conjugation therapeutics: Compounds that interfere with relaxase function could be developed as adjuvants to antibiotics, limiting resistance spread during treatment.

• Identifying resistance reservoirs: Knowledge of conjugation mechanisms helps identify bacterial reservoirs where resistance genes accumulate and from which they disseminate.

• Engineering beneficial conjugation systems: Modified relaxase systems could be engineered to deliver sensitivity genes to resistant populations, potentially reversing resistance in environmental or clinical settings.

This research direction holds promise for developing innovative approaches to the global antibiotic resistance crisis.

What interdisciplinary approaches might yield new insights into DNA relaxase nicK function in bacterial communities?

Interdisciplinary research approaches offer new perspectives on DNA relaxase nicK function:

• Metagenomics and relaxase diversity: Analyzing relaxase genes across diverse bacterial communities can reveal evolutionary relationships and novel functional variants.

• Systems biology modeling: Integrating relaxase function into models of bacterial gene transfer networks can predict community-level effects of relaxase activity.

• Microfluidics and bacterial community dynamics: Microfluidic systems allow observation of conjugation events in controlled microenvironments, linking relaxase function to community behavior.

• Synthetic biology and engineered communities: Constructing bacterial communities with modified relaxase systems can reveal principles of horizontal gene transfer regulation.

• Ecological approaches: Studying how relaxase-mediated gene transfer influences bacterial adaptation in natural environments connects molecular mechanisms to ecological outcomes.

• Computational phylogenetics: Evolutionary analysis of relaxase sequences across species can identify conserved functional domains and species-specific adaptations.

These interdisciplinary approaches connect molecular mechanisms to bacterial community dynamics, evolution, and ecology, providing a more comprehensive understanding of relaxase function in natural and engineered systems.