Recombinant Oceanobacillus iheyensis Tyrosine recombinase XerD (xerD)

What is Oceanobacillus iheyensis XerD and what is its primary function?

Oceanobacillus iheyensis XerD (UniProt: Q7ZAM3) is a tyrosine recombinase enzyme involved in site-specific DNA recombination. It belongs to the family of tyrosine recombinases that catalyze recombination between specific DNA sites . The primary function of XerD in bacteria is to participate in chromosome dimer resolution, a critical process that ensures proper chromosome segregation during cell division .

In most bacterial systems, chromosome dimers form during homologous recombination between circular sister chromosomes during DNA replication. These dimers must be resolved into monomers before cell division can occur. The XerD recombinase, often working cooperatively with XerC in many bacteria, catalyzes site-specific recombination at the dif site located at the junction of the two replication arms . This recombination process is typically coupled to cell division through interaction with the septal protein FtsK .

How does the structure of Oceanobacillus iheyensis XerD compare to other tyrosine recombinases?

Oceanobacillus iheyensis XerD shares structural conservation with other tyrosine recombinases, particularly in its catalytic domain architecture. Crystal structure analyses of tyrosine recombinases have revealed a conserved catalytic (CAT) domain fold across various members of this family .

The XerD protein, like other tyrosine recombinases, contains two major domains:

• Catalytic (CAT) domain: This domain contains the active site responsible for the DNA cleavage and ligation activities. The CAT domain includes the conserved catalytic tetrad R-H-R-Y (arginine-histidine-arginine-tyrosine) essential for the recombination reaction .

• Core-binding (CB) domain: This domain is responsible for DNA recognition and binding. Structural comparisons between XerD and Cre recombinase have shown that their CB domains are closely related, with the three central α-helices of these domains being superposable to within 1.44 Å .

What is known about the genome context of xerD in Oceanobacillus iheyensis?

Oceanobacillus iheyensis HTE831 possesses a 3.6 Mb genome that encodes numerous proteins associated with adaptation to highly alkaline and saline environments . The xerD gene is part of this genomic architecture and contributes to the essential DNA metabolism processes in this extremophile.

Unlike some other bacterial systems like Streptococci/Lactococci where the xerS gene is positioned immediately downstream of its recognition site (difSL) , the genomic context of xerD in O. iheyensis follows the more traditional arrangement seen in organisms like E. coli, where the xerC and xerD genes are typically located distant from each other and from their recombination site.

The genomic analysis of O. iheyensis has revealed that this organism belongs to the Bacillus genus backbone, which comprises approximately 350 core genes conserved across Bacillus species . This conservation extends to essential DNA metabolism genes, including those involved in site-specific recombination systems like XerD.

What are the optimal storage conditions for recombinant Oceanobacillus iheyensis XerD protein?

According to product specifications, the stability and shelf life of recombinant Oceanobacillus iheyensis XerD protein depend on several factors including storage state, buffer formulation, storage temperature, and the intrinsic stability of the protein itself . The optimal storage conditions are:

For research applications requiring long-term storage, the lyophilized form offers twice the shelf life of the liquid preparation . To maintain optimal activity of the recombinant protein:

• Avoid repeated freeze-thaw cycles

• Store small aliquots of the protein when in liquid form

• Reconstitute lyophilized protein immediately before use

• Follow buffer recommendations specific to the intended application

The purity of commercially available recombinant XerD is typically >85% as determined by SDS-PAGE analysis , which is suitable for most research applications including biochemical assays, structural studies, and activity measurements.

How can researchers assess the activity of recombinant XerD in vitro?

Assessing the catalytic activity of recombinant Oceanobacillus iheyensis XerD can be accomplished through several established methodologies:

• Site-specific recombination assays: Researchers can design substrate plasmids containing the dif recognition sequence for XerD. The recombination activity can be monitored by transformation of these plasmids into bacteria expressing XerD, followed by analysis of recombination products .

• Excision assays: Similar to those developed for E. coli XerCD system, these assays involve constructing a cassette with two directly repeated dif sites flanking a selectable marker (such as a kanamycin resistance gene). The excision frequency can be determined by measuring the loss of the selectable marker over time .

• Intermolecular recombination assays: These involve testing recombination between a chromosomally located dif site and a second dif site on a non-replicative plasmid .

• DNA binding assays: Electrophoretic mobility shift assays (EMSA) can be employed to assess the ability of XerD to bind its target DNA sequence.

• In vitro strand exchange assays: Using purified XerD protein and synthetic DNA substrates containing dif sites to directly observe the DNA cleavage and strand exchange reactions.

When designing these assays, it's important to consider that the optimal reaction conditions for O. iheyensis XerD may reflect the alkaliphilic and halotolerant nature of the source organism , potentially requiring buffers with higher pH and salt concentrations than those used for mesophilic recombinases.

What experimental approaches can be used to study XerD-DNA interactions?

Studying the interactions between Oceanobacillus iheyensis XerD and its DNA substrates requires sophisticated biochemical and biophysical techniques:

When designing these experiments, researchers should consider the specific sequence of the DNA substrate. Based on insights from related recombinases, the DNA target likely consists of two imperfect inverted repeats separated by a 6-8 bp central sequence , similar to other tyrosine recombinase recognition sites.

How does the function of O. iheyensis XerD compare with unconventional Xer recombination systems in other bacteria?

The Xer recombination system shows interesting variations across bacterial species, with O. iheyensis XerD representing a more conventional system compared to some specialized adaptations found in other bacteria:

• Conventional XerCD system (including O. iheyensis):

  • Utilizes two distinct recombinases (XerC and XerD)

  • Both recombinases act on a specific dif site

  • Genes encoding XerC and XerD are typically located far from each other and distant from the dif site

  • The process is regulated by the FtsK DNA translocase

• Unconventional XerS/difSL system (Streptococci/Lactococci):

  • Uses a single recombinase (XerS) rather than the XerC/XerD pair

  • Acts on a specific 31-bp sequence called difSL

  • The xerS gene is located immediately upstream of the difSL site

  • This system can functionally substitute for XerCD/dif in E. coli for chromosome dimer resolution

Functional comparison experiments have demonstrated that while these systems differ in their components, they serve the same essential biological function of chromosome dimer resolution. When the XerS/difSL system from Lactococci was introduced into E. coli lacking its native XerCD system, it successfully resolved chromosome dimers, restored normal cell morphology, and provided a growth advantage similar to that of the native XerCD system .

This functional conservation despite mechanistic differences highlights the evolutionary importance of chromosome dimer resolution systems and suggests that O. iheyensis XerD, as part of a conventional XerCD system, plays the same critical role in maintaining genome stability.

What structural features are conserved between O. iheyensis XerD and other tyrosine recombinases?

Structural conservation among tyrosine recombinases, including O. iheyensis XerD, extends across both domains of these proteins:

These structural similarities provide a framework for understanding the function of O. iheyensis XerD through comparison with better-characterized recombinases. The conservation suggests that insights gained from studies of one tyrosine recombinase can often be applied to others, including O. iheyensis XerD, with appropriate consideration of species-specific adaptations.

How might the extreme environment of O. iheyensis influence the properties of its XerD recombinase?

Oceanobacillus iheyensis is an alkaliphilic and extremely halotolerant organism isolated from deep-sea sediment . These extreme environmental adaptations likely influence the properties of all its proteins, including XerD:

• pH adaptation: As an alkaliphilic organism, O. iheyensis thrives in high pH environments. This suggests that its XerD likely has optimal activity at higher pH values compared to recombinases from neutrophilic bacteria. This adaptation could involve:

  • Modified surface charge distribution

  • Altered pKa values of catalytic residues

  • Structural stabilization mechanisms effective at high pH

• Salt tolerance: The extreme halotolerance of O. iheyensis indicates that its XerD may maintain stability and activity at salt concentrations that would denature proteins from non-halotolerant organisms. Potential adaptations include:

  • Increased proportion of acidic residues on the protein surface

  • Enhanced hydrophobic interactions in the protein core

  • Specific ion-binding sites that stabilize the protein structure

• Temperature considerations: Deep-sea environments typically have stable, cold temperatures. The XerD from O. iheyensis might display:

  • Activity over a narrower temperature range

  • Structural adaptations for flexibility at lower temperatures

  • Different energy requirements for conformational changes during catalysis

The genome of O. iheyensis encodes many proteins potentially associated with regulation of intracellular osmotic pressure and pH homeostasis . The XerD recombinase would need to function efficiently within this specialized cellular environment, likely requiring adaptations not present in recombinases from organisms living in less extreme conditions.

How can O. iheyensis XerD be utilized in synthetic biology applications?

Tyrosine recombinases have proven valuable in synthetic biology due to their site-specific DNA manipulation capabilities. O. iheyensis XerD offers several potential applications in this field:

• Genome engineering tools: The site-specific recombination activity of XerD can be harnessed for precise DNA integration, deletion, or inversion in synthetic systems. Its specificity can allow targeted modifications while minimizing off-target effects.

• Memory devices in synthetic circuits: Recombinases can function as biological "switches" that permanently record transient signals through DNA rearrangements. O. iheyensis XerD could serve as a component in synthetic genetic circuits that record environmental or cellular states.

• Protein evolution platforms: The recombination activity can be utilized to generate protein diversity through domain shuffling or directed evolution approaches.

• Assembly of large DNA constructs: Site-specific recombination can facilitate the precise assembly of large DNA fragments, enabling the construction of synthetic chromosomes or complex genetic circuits.

• Extremophilic applications: Given the source organism's adaptation to alkaline and high-salt environments, O. iheyensis XerD might offer unique advantages for synthetic biology applications in extreme conditions where conventional enzymes would be unstable.

When developing such applications, researchers should consider engineering the recognition site specificity through directed evolution or rational design approaches, similar to what has been done with other tyrosine recombinases like Cre and FLP. The potential for cross-reactivity with endogenous recombination sites should also be evaluated when using this system in different host organisms.

What techniques can be used to study the evolutionary relationships between XerD from O. iheyensis and other tyrosine recombinases?

Understanding the evolutionary relationships between O. iheyensis XerD and other tyrosine recombinases requires a multi-faceted approach:

• Phylogenetic analysis:

  • Construction of maximum likelihood or Bayesian phylogenetic trees

  • Analysis of conserved domains across diverse bacterial species

  • Identification of orthologous and paralogous relationships

• Comparative genomics:

  • Analysis of synteny and gene neighborhoods

  • Identification of horizontal gene transfer events

  • Comparison of GC content and codon usage patterns

• Structural bioinformatics:

  • Homology modeling based on crystal structures of related recombinases

  • Comparison of protein folding patterns and conservation of critical residues

  • Analysis of structural adaptations to extreme environments

• Functional complementation studies:

  • Testing whether O. iheyensis XerD can functionally replace XerD in other species

  • Assessing activity under varying conditions (pH, salt, temperature)

  • Evaluation of substrate specificity across different recombinases

Comparative analyses have shown that the backbone of the Bacillus genus is composed of approximately 350 conserved genes , and tyrosine recombinases like XerD are often part of this core genome. Studying how O. iheyensis XerD relates to recombinases from other extremophiles versus mesophilic bacteria can provide insights into how these essential enzymes adapt to different environmental pressures while maintaining their critical cellular functions.

What are the major challenges in expressing and purifying functional O. iheyensis XerD for structural studies?

Obtaining high-quality recombinant O. iheyensis XerD for structural studies presents several challenges that researchers must address:

• Expression optimization:

  • Codon optimization for the expression host

  • Selection of appropriate expression systems (bacterial, yeast, insect, or mammalian)

  • Optimization of induction conditions considering the alkaliphilic origin of the protein

  • Testing different fusion tags (His, GST, MBP) to enhance solubility

• Solubility challenges:

  • DNA-binding proteins like recombinases often have solubility issues

  • Optimization of buffer conditions to reflect the native alkaline and high-salt environment

  • Addition of solubility enhancers or stabilizing agents

  • Exploration of refolding protocols if inclusion bodies form

• Purification considerations:

  • Development of multi-step purification protocols

  • Removal of bound nucleic acids that may co-purify

  • Maintenance of protein activity throughout purification

  • Achievement of high purity (>95%) required for crystallization

• Structural study-specific requirements:

  • For X-ray crystallography: Obtaining diffraction-quality crystals

  • For NMR studies: Isotope labeling and concentration optimization

  • For cryo-EM: Sample homogeneity and concentration requirements

• Activity verification:

  • Development of robust activity assays

  • Ensuring that the purified protein retains its DNA binding and recombination functions

  • Comparison of activity parameters with native enzyme

Current commercial preparations of recombinant O. iheyensis XerD have achieved purities of >85% as determined by SDS-PAGE , which is sufficient for many biochemical applications but may require further purification for high-resolution structural studies.

What unresolved questions remain about the role of XerD in extremophilic bacteria like O. iheyensis?

Despite advances in our understanding of tyrosine recombinases, several important questions remain unanswered regarding XerD in extremophiles like Oceanobacillus iheyensis:

• Adaptation mechanisms:

  • How does O. iheyensis XerD maintain activity in alkaline and high-salt environments?

  • What structural adaptations distinguish it from XerD in non-extremophiles?

  • Are there differences in the kinetics of DNA binding and catalysis compared to mesophilic counterparts?

• Regulatory networks:

  • How is XerD expression regulated in O. iheyensis?

  • Are there extremophile-specific regulatory factors that control XerD activity?

  • How does the FtsK-XerD interaction function in an extremophilic context?

• Recognition site specificity:

  • What is the exact sequence of the dif site in O. iheyensis?

  • How does the recognition specificity compare with other Bacillus species?

  • Are there additional XerD-dependent recombination sites in the O. iheyensis genome?

• Physiological roles:

  • Beyond chromosome dimer resolution, does XerD serve additional functions in O. iheyensis?

  • How does chromosome segregation occur under extreme conditions?

  • Are there interactions with other DNA metabolism proteins specific to extremophiles?

• Evolutionary considerations:

  • Did the XerD system in O. iheyensis evolve independently to function in extreme environments?

  • Or was the pre-existing system adaptable enough to function across diverse conditions?

Addressing these questions would provide valuable insights into both the fundamental biology of tyrosine recombinases and the adaptation strategies employed by extremophilic organisms.

How might comparative studies between O. iheyensis XerD and streptococcal XerS advance our understanding of tyrosine recombinase evolution?

Comparative studies between the conventional XerD system in O. iheyensis and the unconventional XerS system in Streptococci could yield significant insights into the evolution and diversification of tyrosine recombinases:

• Evolutionary transitions:

  • Determining whether the XerCD dual recombinase system or the XerS single recombinase system is ancestral

  • Identifying evolutionary pressures that might drive transitions between these systems

  • Mapping the distribution of different Xer systems across bacterial phylogeny

• Functional convergence:

  • Understanding how different recombinase systems evolved to perform the same essential function

  • Identifying the minimal components required for site-specific recombination

  • Determining whether there are functional advantages to either system in different contexts

• Mechanistic comparisons:

  • Analyzing how a single recombinase (XerS) can accomplish what typically requires two cooperating recombinases (XerC and XerD)

  • Comparing the catalytic efficiency and fidelity of both systems

  • Investigating differences in regulation and interaction with other cellular components

• Structure-function relationships:

  • Identifying structural adaptations that allow XerS to function independently

  • Comparing DNA binding domains and their recognition specificities

  • Analyzing the evolution of protein-protein interaction interfaces

Such comparative studies could reveal fundamental principles of molecular evolution, including how essential cellular functions can be maintained despite significant changes in the underlying molecular machinery. The fact that the XerS/difSL system can functionally substitute for XerCD/dif in E. coli provides a foundation for these investigations by demonstrating the functional equivalence of these divergent systems.