Recombinant Vibrio vulnificus Tyrosine recombinase XerD (xerD)

What is the function of XerD recombinase in Vibrio vulnificus and how does it compare to XerD in other bacteria?

V. vulnificus XerD, like its homologs in other bacteria, is primarily involved in chromosome dimer resolution, which is essential for proper chromosome segregation during cell division. The Xer site-specific recombination system resolves circular chromosome dimers formed by homologous recombination during DNA replication.

In E. coli, this system consists of two paralogous tyrosine recombinases, XerC (298 aa) and XerD (298 aa), which act on a 28 bp DNA sequence called the dif site located in the termination region of the chromosome . The V. vulnificus XerD likely follows a similar functional pattern, though with species-specific variations in protein sequence and dif site recognition.

Comparative analysis with other Vibrio species, particularly V. cholerae (where XerC/D are well-studied), suggests that V. vulnificus XerD may also be involved in the integration of mobile genetic elements, as observed in the CTXφ, VGJφ, and TLCφ phages that exploit the Xer system in V. cholerae .

How do XerC and XerD cooperate in bacterial chromosome maintenance?

In two-recombinase systems like that of E. coli and likely V. vulnificus, XerC and XerD act cooperatively to perform site-specific recombination at dif sites. The synaptic XerCD/dif complex consists of two XerC and two XerD subunits respectively bound to two dif sites . This tetrameric arrangement allows for the coordinated exchange of DNA strands.

The recombination process follows these steps:

• XerC and XerD bind to their respective recognition sequences within the dif site

• The proteins bring two dif sites together to form a synaptic complex

• One recombinase initiates strand cleavage and exchange

• The second recombinase completes the recombination process

This cooperation is highly regulated and often involves interaction with the cell division protein FtsK, which helps activate the recombination at the appropriate time during cell division . Structural studies of Xer synaptic complexes have revealed that activation of DNA strand cleavage and rejoining involves large conformational changes and DNA bending .

What is the dif site in V. vulnificus and how is it recognized by XerD?

While the specific sequence of the V. vulnificus dif site is not detailed in the provided sources, we can infer its characteristics based on dif sites in related bacteria. In general, bacterial dif sites are approximately 28-30 bp sequences comprising:

• A XerC-binding region

• A central region (6-8 bp)

• A XerD-binding region

The specific sequence of the dif site can vary between bacterial species, but the functional architecture remains conserved. In H. pylori, for example, the XerH-difH system shows how small sequence asymmetry in dif defines protein conformation in the synaptic complex and orchestrates the order of DNA strand exchanges .

XerD recognizes and binds to its specific half of the dif site through its DNA-binding domain. This recognition is sequence-specific and involves contacts between amino acid residues and nucleotide bases. Structural studies of Xer recombinases have revealed a conserved catalytic domain fold that facilitates DNA binding and cleavage .

How does the XerD/XerC system resolve chromosome dimers in bacteria?

The Xer system resolves chromosome dimers through a precise multi-step process:

• Recognition and binding: XerC and XerD recognize and bind to their respective halves of the dif sites on the chromosome dimer.

• Synaptic complex formation: Two dif sites are brought together to form a tetrameric complex containing two XerC and two XerD molecules.

• First strand exchange: Typically, XerD makes the first cleavage, using its catalytic tyrosine residue to attack the phosphodiester bond at a specific position in the DNA backbone, forming a covalent 3'-phosphotyrosyl link and releasing a free 5'-hydroxyl group.

• Strand migration: The free 5'-hydroxyl attacks the phosphotyrosyl link of the partner dif site, resulting in strand exchange.

• Second strand exchange: XerC then performs a similar reaction on the second pair of strands.

• Resolution: The result is two separate circular chromosomes, each containing one dif site.

This process is highly regulated and often depends on the cell division protein FtsK, which can activate XerD catalysis by inducing conformational changes in the synaptic complex . The process ensures that chromosome dimers are resolved before cell division completes, allowing proper segregation of genetic material to daughter cells.

What structural insights have been gained about Xer recombinases and how might they apply to V. vulnificus XerD?

Recent structural studies, particularly on H. pylori XerH (a single recombinase system), have provided valuable insights into Xer recombination mechanisms that may apply to V. vulnificus XerD. High-resolution crystal structures of XerH-difH synaptic complexes have been obtained, representing pre-cleavage and post-cleavage intermediates in the recombination pathway .

Key structural insights include:

• Initial assembly in inactive state: XerH-difH synaptic complexes initially assemble with straight DNA in an inactive state.

• Conformational activation: A major conformational change is required for catalytic activation, involving protein-protein interface rearrangements and DNA bending.

• Asymmetric recognition: Small differences in the arms of the dif recombination sites can choreograph the reaction steps.

• FtsK-mediated activation: The conformational change may be promoted by interaction with FtsK, which licenses the reaction at the appropriate time during cell division.

These structural features likely apply to V. vulnificus XerD given the conserved nature of tyrosine recombinase mechanisms. Modeling based on these structures can provide insights into V. vulnificus XerD function, as demonstrated by the extension of structural findings to the E. coli XerC/D-dif system .

How does the FtsK protein interact with and regulate XerD activity in bacteria?

FtsK is a DNA translocase associated with the bacterial cell division machinery that plays a crucial role in activating Xer recombination. While specific details for V. vulnificus are not provided in the search results, the general mechanism based on other bacterial systems involves:

• Directional DNA translocation: FtsK loads onto chromosomal DNA near the division septum and translocates toward the dif site.

• XerD activation: Upon reaching the Xer-dif complex, FtsK interacts directly with XerD, likely through its C-terminal domain.

• Conformational change induction: This interaction promotes a conformational change in the Xer-dif complex, switching it from an inactive state with straight DNA to an active state with bent DNA.

• Catalytic activation: The conformational change positions the catalytic residues of XerD appropriately for DNA cleavage, initiating the recombination process.

Structural studies have revealed that "bending of the dif sites does not occur concomitantly with synaptic complex assembly but at a post-synaptic step, when the accessory factor FtsK might be needed to license the reaction by promoting a conformational change" . This regulated activation ensures that chromosome dimer resolution occurs at the appropriate time and place during cell division.

What are the key catalytic residues in tyrosine recombinases like XerD and how can they be studied through mutagenesis?

Tyrosine recombinases, including XerD, share a conserved catalytic mechanism with key residues essential for DNA cleavage and rejoining. While specific residues in V. vulnificus XerD are not detailed in the provided sources, we can infer them based on other tyrosine recombinases:

Key catalytic residues typically include:

• Catalytic tyrosine (nucleophile for DNA cleavage)

• Conserved basic residues (typically arginine and lysine) for stabilizing the phosphate transition state

• Histidine or other residues that may act as a general base

Mutagenesis approaches for studying these residues include:

By systematically mutating these residues and analyzing the effects on recombination activity, researchers can elucidate the precise roles of each residue in the catalytic mechanism of V. vulnificus XerD.

How do phages exploit the Xer recombination system in Vibrio species, and could similar mechanisms exist in V. vulnificus?

Phages have evolved sophisticated mechanisms to exploit bacterial Xer recombination systems for integration into host genomes. In V. cholerae, several phages hijack the XerC/D system:

• CTXφ (Cholera Toxin Phage): This filamentous phage encodes the cholera toxin and integrates through a unique mechanism involving its single-stranded DNA form. It forms a "∼150 bp folded structure created by the intra-strand base pairing interaction between two palindromic attP sites (attP1 and attP2) separated by 90 nt on the ssDNA sequence" . The attP sites resemble the XerC side of dif sites but differ on the XerD side, limiting catalysis to XerC.

• VGJφ (Vibrio Guillermo Javier filamentous phage) and TLCφ (Toxic Linked Cryptic): These phages also contain dif-like attachment sites (attP) that serve as substrates for the Xer recombination system .

Given the evolutionary relationships between Vibrio species, it is plausible that similar phage integration mechanisms might exist in V. vulnificus. Identifying such mechanisms would involve:

• Genomic analysis to identify integrated prophages with sequences resembling dif sites at their junctions

• Experimental verification of XerC/D-mediated integration

• Characterization of the specific attP sites and the mechanism of integration

Understanding these mechanisms could provide insights into horizontal gene transfer in V. vulnificus and potentially reveal new tools for genetic engineering based on site-specific recombination systems.

What are optimal strategies for cloning and expressing recombinant V. vulnificus XerD?

Based on successful approaches used for other Xer recombinases, the following strategies would be optimal for cloning and expressing recombinant V. vulnificus XerD:

Gene synthesis and optimization:

• Synthesize the xerD gene with codon optimization for the expression host (typically E. coli)

• Include appropriate restriction sites for cloning flexibility

• Consider the approach used for H. pylori XerH: "Full-length xerH from H. pylori strain 26995 was synthesized with codon-optimization for over-expression in E. coli"

Expression vector selection:

• Choose vectors with strong inducible promoters (T7, tac, etc.)

• Include affinity tags for purification (His-tag, GST, etc.)

• Consider vectors that provide fusion partners to enhance solubility

Expression conditions:

• Test multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express, etc.)

• Optimize induction parameters: temperature (often lowered to 16-18°C for better folding), inducer concentration, and induction time

• Consider autoinduction media for gentler protein expression

Solubility enhancement:

• Include solubility-enhancing additives in the growth media or lysis buffer

• Test different fusion partners if initial constructs show poor solubility

• Consider co-expression with chaperones if misfolding is an issue

Purification strategy:

• Design a multi-step purification scheme:

  • Initial capture by affinity chromatography (Ni-NTA for His-tagged proteins)

  • Intermediate purification by ion exchange chromatography

  • Final polishing by size exclusion chromatography

• Include protease inhibitors throughout purification

• Optimize buffer conditions to maintain protein stability and activity

This approach, adapted from successful expression of other recombinases, should yield pure, active V. vulnificus XerD suitable for biochemical and structural studies.

How can in vitro recombination assays be designed to study V. vulnificus XerD activity?

In vitro recombination assays for V. vulnificus XerD would need to mimic the natural reaction conditions while allowing for convenient detection and quantification of recombination products. Based on approaches used for other Xer recombinases, the following assays could be developed:

Substrate preparation:

• Synthesize DNA fragments containing the V. vulnificus dif site

• Create substrates with different configurations: linear fragments, supercoiled plasmids, or oligonucleotide substrates

• Include fluorescent or radioactive labels for detection purposes

Basic recombination assay:

• Incubate purified XerD (and XerC if using a two-recombinase system) with dif-containing substrates

• Include appropriate buffer conditions (typically containing divalent cations like Mg²⁺)

• Analyze products by gel electrophoresis (agarose or polyacrylamide)

• Detect products by fluorescence scanning, autoradiography, or ethidium bromide staining

Suicide substrate assay:

• Design substrates that trap reaction intermediates (e.g., with nicks or non-cleavable bonds)

• Useful for isolating and characterizing protein-DNA covalent complexes

• Can determine which strand is cleaved first and identify the active recombinase

Kinetic analysis:

• Monitor reaction progress over time by taking samples at different time points

• Determine reaction rates under different conditions

• Analyze the effect of protein concentration, temperature, pH, and salt concentration

Inhibitor screening:

• Test potential inhibitors by including them in the reaction mixture

• Quantify the degree of inhibition under different conditions

• Useful for identifying potential antimicrobial compounds

Additional considerations:

• Include controls: inactive mutants (e.g., catalytic tyrosine mutants), no-enzyme controls

• For two-recombinase systems, test the requirement for both recombinases

• Consider the potential requirement for accessory factors like FtsK

These assays would provide comprehensive insights into the biochemical properties and catalytic mechanism of V. vulnificus XerD.

What techniques are available for studying XerD-DNA interactions at the molecular level?

Several sophisticated techniques can be employed to study the molecular interactions between V. vulnificus XerD and its DNA substrates:

Electrophoretic Mobility Shift Assay (EMSA):

• Incubate purified XerD with labeled DNA fragments containing dif sites

• Analyze complexes by native gel electrophoresis

• Determine binding affinity, specificity, and stoichiometry

• Can be used competitively to identify specific binding sequences

Footprinting Techniques:

• DNase I footprinting: Identifies regions of DNA protected by bound protein

• Hydroxyl radical footprinting: Provides higher resolution of protein-DNA contacts

• DMS footprinting: Reveals specific guanine contacts in the major groove

X-ray Crystallography:

• Determine high-resolution structures of XerD-DNA complexes

• Similar to the approach used for H. pylori XerH: "two high-resolution structures of Helicobacter pylori XerH with its recombination site DNA difH, representing pre-cleavage and post-cleavage synaptic intermediates"

• Reveals precise atomic interactions between protein residues and DNA bases

Cryo-Electron Microscopy (Cryo-EM):

• Alternative approach for structural determination, especially useful for larger complexes

• Can capture different conformational states during the recombination process

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Useful for studying dynamics of protein-DNA interactions in solution

• Can provide information about conformational changes upon DNA binding

Surface Plasmon Resonance (SPR):

• Real-time measurement of binding kinetics and affinity

• Allows determination of association and dissociation rates

Fluorescence-based Techniques:

• Fluorescence anisotropy to measure binding affinities

• Förster resonance energy transfer (FRET) to detect conformational changes

• Fluorescence lifetime measurements for detailed binding characterization

Atomic Force Microscopy (AFM):

• Visualize XerD-DNA complexes at the single-molecule level

• Observe topological changes in DNA upon protein binding

Single-molecule Techniques:

• Optical or magnetic tweezers to study mechanical aspects of recombination

• Similar to approaches mentioned: "single molecule fluorescence studies of XerC/D-dif recombination"

These techniques, often used in combination, would provide comprehensive insights into how V. vulnificus XerD recognizes, binds, and catalyzes recombination at dif sites.

How can the recombination activity of XerD be monitored in real-time?

Real-time monitoring of XerD recombination activity provides valuable insights into reaction kinetics and mechanisms. Several approaches can be adapted from existing methodologies:

Fluorescence-based assays:

• Design DNA substrates with fluorophore-quencher pairs that change signal upon recombination

• Position fluorescent probes so that FRET (Förster Resonance Energy Transfer) signals change during recombination

• Monitor changes in fluorescence intensity or anisotropy in real-time using a fluorescence plate reader or spectrofluorometer

Real-time PCR-based detection:

• Design recombination substrates that alter primer binding sites or amplicon length upon recombination

• Use quantitative PCR to monitor the appearance of recombination products over time

• This approach could adapt elements from real-time RPA (Recombinase Polymerase Amplification) methods described for V. vulnificus detection

Surface-based detection systems:

• Immobilize DNA substrates on biosensor surfaces

• Monitor recombination events using surface plasmon resonance (SPR) or biolayer interferometry (BLI)

• These methods provide real-time binding kinetics and can detect conformational changes

Single-molecule techniques:

• Use fluorescence microscopy to observe individual recombination events in real-time

• Apply techniques like Total Internal Reflection Fluorescence (TIRF) microscopy to reduce background

• This approach has been successful for studying XerC/D-dif recombination: "single molecule fluorescence studies of XerC/D-dif recombination"

Tethered particle motion (TPM):

• Attach one end of the DNA substrate to a surface and the other to a bead

• Monitor changes in bead movement as recombination alters DNA topology

• Provides real-time information about individual recombination events

Electrochemical detection:

• Design DNA substrates with electrochemical tags that generate signals upon recombination

• Monitor changes in current or potential in real-time

Continuous gel electrophoresis:

• Perform recombination reactions in a continuous-flow system coupled to gel electrophoresis

• Sample and analyze the reaction mixture at defined intervals

For implementing these techniques with V. vulnificus XerD, researchers should consider the specific properties of the recombination system, including potential requirements for XerC, the dif site sequence, and accessory factors like FtsK.

How might understanding V. vulnificus XerD contribute to developing new antimicrobial strategies?

Understanding V. vulnificus XerD recombinase could lead to novel antimicrobial strategies through several approaches:

Direct inhibition of essential function:

• Xer recombinases are essential for chromosome maintenance in bacteria

• Specific inhibitors of XerD could prevent chromosome dimer resolution, leading to segregation defects and cell death

• This approach would leverage the "systems biological approach that efficiently utilizes genomic information for drug targeting and discovery"

Structure-based drug design:

• High-resolution structures of Xer recombinases, like those obtained for XerH , provide templates for rational drug design

• Virtual screening against the active site or protein-protein interaction interfaces could identify lead compounds

• Fragment-based approaches could identify small molecules that disrupt XerD function

Targeting species-specific features:

• Identifying unique structural or functional features of V. vulnificus XerD could allow for species-specific targeting

• This could lead to narrow-spectrum antimicrobials with fewer off-target effects on beneficial microbiota

Metabolic network analysis:

• Integration with metabolic models like "VvuMBEL943" could provide context for XerD inhibition within the broader cellular network

• This systems approach could identify synergistic drug combinations targeting both XerD and related metabolic pathways

Phage-inspired strategies:

• Understanding how phages exploit the Xer system could inspire biomimetic approaches

• Engineered phage components could be developed to target and disrupt V. vulnificus XerD function

Blocking horizontal gene transfer:

• If V. vulnificus XerD is involved in acquisition of mobile genetic elements (similar to V. cholerae ), inhibitors could reduce acquisition of virulence factors or antimicrobial resistance genes

Disrupting pathogenesis:

• If XerD plays a role in stress response or virulence gene expression, targeting it could attenuate pathogenicity

Developing these approaches would require thorough validation of XerD as an antimicrobial target, including demonstration of its essentiality in V. vulnificus and careful assessment of potential resistance mechanisms.

What potential applications exist for using Xer recombinases in biotechnology or genetic engineering?

Xer recombinases, including V. vulnificus XerD, have significant potential in biotechnology and genetic engineering applications:

Site-specific genome integration tools:

• Xer recombinases could be adapted as alternatives to established systems like Cre/lox or FLP/FRT

• They could provide precise integration of genetic material at defined genomic locations

• This would leverage their natural role in site-specific recombination: "tyrosine recombinases (as exemplified by Cre and Flp) provide powerful genetic engineering tools"

Genetic circuit design:

• Engineered Xer recombination systems could serve as genetic switches in synthetic biology applications

• Inducible recombination could allow for conditional gene expression or deletion

Genome editing in bacteria:

• Xer-based systems could complement CRISPR-Cas tools for bacterial genome engineering

• They could provide seamless deletion or insertion of DNA sequences at specific sites

DNA assembly technologies:

• Site-specific recombination could facilitate in vitro or in vivo assembly of DNA fragments

• This could enable construction of large DNA molecules for synthetic biology applications

Markerless bacterial engineering:

• Similar to "XerH-based genetic engineering tools that have been recently introduced for markerless gene deletions in H. pylori"

• Such systems could be adapted for V. vulnificus and other bacteria, allowing clean genetic modifications without antibiotic resistance markers

Programmable DNA topology manipulation:

• The ability of Xer recombinases to resolve DNA dimers could be harnessed to manipulate DNA topology in vitro

• This could be useful in nanotechnology applications or for studying DNA structure-function relationships

Phage engineering:

• Understanding how phages exploit Xer systems for integration could lead to engineered phages for various applications

• This could include phage therapy, gene delivery, or diagnostic applications

In vitro recombination systems:

• Purified recombinant XerD could be used in in vitro DNA manipulation techniques

• This could include site-specific labeling of DNA molecules or assembly of DNA nanostructures

These applications would require detailed characterization of V. vulnificus XerD specificity, activity, and regulation, as well as engineering efforts to optimize the system for specific biotechnological purposes.

How could inhibition of XerD affect V. vulnificus pathogenesis and survival?

Inhibition of XerD in V. vulnificus would likely have profound effects on bacterial physiology and pathogenesis through several mechanisms:

Disruption of chromosome segregation:

• XerD's primary role in chromosome dimer resolution is essential for proper chromosome segregation

• Inhibition would lead to accumulation of chromosome dimers, causing cell division defects

• This would result in filamentation, growth arrest, and eventual cell death

Genomic instability:

• Unresolved chromosome dimers increase genomic instability

• This may lead to increased mutation rates and DNA damage, further compromising bacterial fitness

Stress response alteration:

• Bacteria with compromised Xer systems would likely activate stress response pathways

• This could affect multiple aspects of bacterial physiology, including virulence gene expression

Reduced environmental persistence:

• V. vulnificus survival in various environmental conditions could be compromised

• This might reduce contamination of shellfish and other sources of human infection

Impaired biofilm formation:

• Cell division defects would likely impact biofilm structure and formation

• Biofilms are important for environmental persistence and resistance to antibiotics and host defenses

Attenuated virulence:

• Growth defects would directly impact the ability of V. vulnificus to proliferate during infection

• Changes in gene expression due to chromosome segregation stress might alter virulence factor production

Synergy with host defenses:

• Bacteria with compromised XerD function might be more susceptible to host immune defenses

• This could include increased sensitivity to oxidative stress generated by neutrophils

Potential impact on horizontal gene transfer:

• If V. vulnificus XerD is involved in the integration of mobile genetic elements (as seen in V. cholerae ), inhibition could reduce acquisition of new virulence or resistance genes

These effects make XerD an attractive potential target for antimicrobial development against V. vulnificus, particularly given its essential role in bacterial chromosome maintenance and the absence of a direct homolog in human cells.

What role might XerD play in horizontal gene transfer and antimicrobial resistance in V. vulnificus?

Based on evidence from related Vibrio species, XerD may play significant roles in horizontal gene transfer (HGT) and consequently in antimicrobial resistance (AMR) acquisition in V. vulnificus:

Phage integration:

• In V. cholerae, XerC and XerD are hijacked by vibriophages such as CTXφ, VGJφ, and TLCφ for chromosomal integration

• These Integrative Mobile Elements Exploiting Xer (IMEX) use dif-like attachment sites (attP) for integration

• Similar mechanisms might exist in V. vulnificus, potentially facilitating the acquisition of phage-encoded virulence or resistance genes

Integration of mobile genetic elements:

• The Xer recombination system might mediate integration of other mobile genetic elements beyond phages

• This could include integrative conjugative elements (ICEs) or genomic islands carrying AMR genes

Plasmid stability:

• Xer recombinases resolve plasmid dimers in many bacteria: "Small plasmids, like those in the ColE1 family, use the chromosomally encoded dimer resolution system of their host"

• By ensuring stable plasmid inheritance, XerD could indirectly contribute to maintenance of plasmid-borne AMR genes

Genomic plasticity:

• Xer-mediated recombination might contribute to chromosomal rearrangements

• This could facilitate adaptation to selective pressures, including antibiotics

Integration mechanisms:

• Different integration mechanisms might exist, similar to those observed in V. cholerae:

  • Direct ssDNA integration as seen with CTXφ

  • Integration through different mechanisms depending on phage type (CTXφ-type, VGJφ-type, TLCφ-type)

Co-evolution with mobile elements:

• Mobile genetic elements may have evolved specific mechanisms to exploit the Xer system in V. vulnificus

• These could include unique attP sites or accessory factors that modulate Xer activity

Environmental adaptation:

• Xer-mediated HGT might be particularly important in the aquatic environments where V. vulnificus naturally occurs

• This could facilitate adaptation to changing environmental conditions and acquisition of beneficial genes

Investigation of these potential roles would require:

• Genomic analysis to identify integrated elements with dif-like junction sequences

• Experimental verification of XerD-mediated integration

• Characterization of potential attP sites in V. vulnificus phages and other mobile elements

• Assessment of the impact of XerD inhibition on HGT and AMR acquisition

Understanding these mechanisms could provide insights into the evolution of virulence and antimicrobial resistance in V. vulnificus and inform strategies to combat this pathogen.