Recombinant Bartonella henselae Exodeoxyribonuclease 7 large subunit (xseA)

What is Bartonella henselae exodeoxyribonuclease 7 and what are its biological functions?

Bartonella henselae exodeoxyribonuclease 7 (ExoVII) is a nuclease complex involved in DNA processing pathways. The enzyme participates in multiple nucleic acid-dependent pathways, particularly in DNA repair mechanisms. In Escherichia coli, where ExoVII has been more extensively characterized, it functions in DNA damage repair, mutation avoidance, and mismatch repair pathways . The enzyme is unique among bacterial exonucleases in that it demonstrates both 3′→5′ and 5′→3′ exonuclease activity on single-stranded DNA substrates and operates independently of metal cofactors . In the context of Bartonella species, ExoVII likely plays a role in maintaining genomic integrity during the bacterium's complex lifecycle, which involves both mammalian hosts and arthropod vectors.

How is exodeoxyribonuclease 7 structured and what is the relationship between the large (XseA) and small (XseB) subunits?

Exodeoxyribonuclease 7 consists of two distinct subunits: a large subunit (XseA) and multiple copies of a small subunit (XseB). Recent cryoEM structural studies of E. coli ExoVII revealed a complex quaternary structure with XseA forming two pseudo-twofold symmetric dimers that create a protein catenane through reciprocal catalytic domain and N-terminal OB-fold swapping events . Each XseA subunit is associated with multiple XseB protomers (approximately six per XseA), which form a stiff, linear array extending between the XseA nuclease and C-terminal domains .

The XseA subunit contains:

• An N-terminal OB-fold domain predicted to bind single-stranded DNA

• A catalytic nuclease domain

• An extended helical segment that interacts with XseB

• A C-terminal domain with a β-barrel structure

The XseB subunit primarily consists of an α-helical hairpin structure (approximately 80 residues in length) that forms three-helix bundles with other XseB subunits . Importantly, the XseB subunit closest to the XseA catalytic domain contributes to completing the nuclease fold, suggesting an essential role in enzyme activity beyond simply structural support.

How conserved is the XseA protein across Bartonella species compared to other bacterial genera?

Comparative genomic analyses indicate that XseA is highly conserved across Bartonella species, with significant nucleotide identity between B. henselae strains. While the search results don't provide specific sequence identity percentages between Bartonella XseA and those of other bacteria, studies of related proteins like P26 in Bartonella show significant sequence conservation within the genus and moderate conservation with orthologs in related alpha-proteobacteria such as Brucella and Agrobacterium .

The phylogenetic analysis of ExoVII components indicates that the length of ExoVII homologs changes in quantized steps corresponding to the addition or removal of one XseB subunit and an associated segment of XseA , suggesting evolutionary pressure to maintain specific stoichiometric relationships between the subunits across bacterial species.

What expression systems are optimal for producing functional recombinant B. henselae XseA?

Based on the search results and research practices for similar Bartonella proteins, several expression systems have proven effective for producing recombinant Bartonella proteins:

• Baculovirus expression system: This eukaryotic expression system has been successfully used for the production of recombinant B. henselae exodeoxyribonuclease 7 small subunit (XseB) , suggesting it may also be suitable for XseA production.

• E. coli expression systems: For other Bartonella proteins such as P26, E. coli expression systems using vectors like pMX (a modified pGEX-2T vector) have been effective . These systems often produce recombinant proteins as fusions with glutathione S-transferase (GST), which can be cleaved post-expression.

When expressing XseA, researchers should consider:

• Codon optimization for the expression host

• Addition of affinity tags (His-tag, GST) for purification

• Selection of appropriate promoters for controlled expression

• Co-expression with XseB if functional activity is required, as the small subunit contributes to the nuclease domain structure

What purification strategies are most effective for isolating recombinant B. henselae XseA?

Effective purification of recombinant B. henselae XseA would likely follow strategies used for similar complex proteins:

• Affinity chromatography: Using affinity tags such as His-tags or GST fusions for initial capture.

• Ion exchange chromatography: For further purification based on the protein's charge properties.

• Size exclusion chromatography: As a final polishing step and to analyze the oligomeric state of the protein.

For co-purification of XseA with XseB to obtain the functional holoenzyme, strategies might include:

• Co-expression of both subunits in the same cell

• Sequential affinity purification using different tags on each subunit

• Careful buffer selection to maintain the complex integrity

Research on related proteins like B. henselae P26 employed GST fusion protein expression followed by thrombin cleavage to release the target protein from GST, with subsequent concentration steps . Similar approaches could be adapted for XseA, with consideration for its larger size and potential complexity.

How can the enzymatic activity of recombinant B. henselae XseA be assessed in vitro?

Assessment of recombinant B. henselae XseA enzymatic activity would likely involve nuclease activity assays similar to those used for other exonucleases:

• DNA substrate degradation assays: Using radiolabeled or fluorescently labeled single-stranded DNA substrates to monitor nuclease activity by:

  • Gel electrophoresis to visualize substrate degradation

  • Fluorescence-based assays measuring the release of labeled nucleotides

  • Real-time monitoring of activity using FRET-labeled substrates

• Direction-specific activity assays: To verify both 3′→5′ and 5′→3′ exonuclease activities:

  • Specially designed DNA substrates with blocked ends

  • Substrates with specific fluorescent labels at either the 5′ or 3′ end

• Cofactor dependency analysis: To confirm the metal cofactor-independent nature of ExoVII:

  • Activity assays in the presence/absence of EDTA

  • Testing with various divalent metal ions to confirm lack of enhancement

For functional studies, it's important to recognize that XseA likely requires association with XseB for full activity, as structural studies of E. coli ExoVII show that XseB contributes to completing the nuclease fold of XseA .

How can recombinant B. henselae XseA be utilized for developing diagnostic tools for bartonellosis?

Recombinant B. henselae XseA presents several potential applications for diagnostic tool development:

• Serological diagnostic assays: Similar to the approach used with B. henselae P26 and Pap31 proteins , recombinant XseA could be evaluated as an antigen for detecting anti-Bartonella antibodies in infected hosts through:

  • Enzyme-linked immunosorbent assays (ELISA)

  • Western blot analysis

  • Immunofluorescence assays

• Molecular diagnostic targets: XseA gene sequences might serve as specific targets for molecular detection of B. henselae:

  • PCR-based detection methods

  • DNA hybridization assays

  • LAMP (Loop-mediated isothermal amplification) assays

Research on B. henselae Pap31 assessed its diagnostic utility for detecting anti-Bartonella antibodies in infected dogs and humans, finding 72% sensitivity and 61% specificity at a cutoff value of 0.215 for human bartonelloses . Similar evaluation would be necessary for XseA to determine its diagnostic potential, recognizing that ideal diagnostic targets should elicit strong, specific antibody responses or contain sequences unique to B. henselae.

What advantages might B. henselae XseA offer as a target for molecular diagnostics compared to other Bartonella proteins?

As a target for molecular diagnostics, B. henselae XseA may offer several potential advantages:

• Genetic conservation with strain-specific variations: If XseA exhibits high conservation across Bartonella species with specific variations between strains (similar to p26 ), it could enable:

  • Species-level identification

  • Strain typing and genotyping of isolates

  • Differentiation between human-associated and animal-associated strains

• Essential cellular function: As an enzyme involved in DNA repair and maintenance, XseA likely experiences different selective pressures than surface antigens, potentially resulting in:

  • More stable genetic sequences less subject to immune selection

  • Lower mutation rates in critical functional domains

  • Consistent expression across different growth conditions

• Potential unique sequences: XseA may contain regions unique to Bartonella that could reduce cross-reactivity with other bacterial species, improving diagnostic specificity.

Research characterizing B. henselae strains in South Korea demonstrated that sequence analysis of specific genes can distinguish between different genotypes (I and II) and correlate with the source of isolation (human versus feline) . Similar analysis of XseA sequences could potentially provide diagnostically useful information about strains and their origin.

How does B. henselae XseA function in DNA repair pathways, and what implications might this have for bacterial pathogenesis?

While the specific role of B. henselae XseA in DNA repair has not been directly characterized in the search results, extrapolation from E. coli ExoVII function suggests:

• DNA damage repair: In E. coli, ExoVII participates in DNA damage repair pathways , suggesting B. henselae XseA likely plays a similar role in:

  • Processing damaged DNA ends

  • Removing nucleotides with damage-induced modifications

  • Preparing DNA ends for repair synthesis

• Mutation avoidance: E. coli ExoVII contributes to mutation avoidance mechanisms , indicating B. henselae XseA may help:

  • Maintain genomic stability during replication

  • Process potentially mutagenic DNA structures

  • Work cooperatively with other repair enzymes (RecJ, ExoI, SbcCD)

• Pathogenesis implications: These DNA repair functions may impact pathogenesis by:

  • Enabling survival within host cells, where the bacterium encounters oxidative stress

  • Maintaining genomic integrity during persistent infections

  • Contributing to host adaptation through controlled mutation rates

In E. coli, studies have shown that combinations of mutations in single-strand DNases (ExoI, RecJ, ExoVII, and SbcCD) affect recombination frequency and UV sensitivity , suggesting these enzymes have overlapping functions in maintaining genomic integrity. In the context of a pathogen like B. henselae that establishes persistent infections, efficient DNA repair mechanisms including XseA would be critical for survival within the host environment.

What domain architecture characterizes B. henselae XseA, and how do individual domains contribute to its function?

Based on structural studies of E. coli ExoVII and sequence analysis, B. henselae XseA likely has a complex domain architecture consisting of:

How does the interaction between XseA and XseB regulate the nuclease activity of the enzyme complex?

Based on structural studies of E. coli ExoVII, the interaction between XseA and XseB appears to regulate nuclease activity through several mechanisms:

• Completion of the nuclease fold: The XseB subunit closest to the XseA catalytic domain contributes a β-strand that docks edgewise against the β-sheet in the XseA catalytic domain, completing the nuclease fold . This structural arrangement suggests that proper XseA-XseB interaction is essential for forming a functional active site.

• Autoinhibition in the DNA-free state: The architectural features of E. coli ExoVII indicate that the enzyme initially adopts an autoinhibited state through steric occlusion, which is relieved when appropriate DNA substrates are encountered . This suggests a regulated activation mechanism dependent on substrate binding.

• DNA-induced conformational changes: The structural analysis suggests that "the dissociation of the complex induced by the binding of DNA substrates may be a strategy to regulate its activity" . This implies that DNA binding triggers conformational changes that alter the XseA-XseB interaction.

In E. coli, the ExoVII complex has been determined to contain four XseA subunits and 24 XseB subunits in a XseA₄·XseB₂₄ stoichiometry , which is significantly different from earlier proposals of XseA₁·XseB₄ or XseA₁·XseB₆ stoichiometries. This complex quaternary structure suggests sophisticated regulation of enzyme assembly and activity that would likely also exist in the B. henselae homolog.

What role might post-translational modifications play in regulating B. henselae XseA activity?

While the search results do not specifically address post-translational modifications (PTMs) of B. henselae XseA, several mechanisms could potentially regulate its activity:

• Phosphorylation: Phosphorylation of key residues could regulate:

  • Protein-protein interactions between XseA and XseB

  • Substrate binding affinity

  • Catalytic activity through conformational changes

• Proteolytic processing: Similar to observations with B. henselae P26, which exists in both preprotein and mature forms , XseA might undergo proteolytic processing:

  • Removal of N-terminal signal sequences

  • Activation through cleavage of inhibitory domains

  • Regulation of cellular localization

• Redox-based regulation: Given the role of ExoVII in DNA repair, its activity might be modulated by the cellular redox state:

  • Oxidation/reduction of cysteine residues

  • Formation/disruption of disulfide bonds

  • Responses to oxidative stress conditions

The complex quaternary structure of ExoVII, with its unique catenane-like arrangement and extensive subunit interactions , provides numerous potential sites where PTMs could regulate assembly, stability, or activity of the enzyme complex. Investigation of these regulatory mechanisms would require targeted proteomic approaches to identify PTMs and functional studies to determine their effects on enzyme activity.

What expression vector and host cell combinations yield optimal production of soluble recombinant B. henselae XseA?

Based on the search results and approaches used for other Bartonella proteins, several expression systems could be considered for optimal production of soluble recombinant B. henselae XseA:

• E. coli expression systems:

  • Vectors: pET-based vectors (e.g., pET200D/TOPO) have been successfully used for Bartonella proteins

  • Host strains: BL21(DE3) is commonly used for protein expression

  • Tags: N-terminal His-tags or GST fusions can improve solubility and facilitate purification

  • Considerations: Codon optimization may be necessary due to differences in codon usage between Bartonella and E. coli

• Baculovirus expression system:

  • Used successfully for B. henselae XseB

  • May provide better folding environment for complex multidomain proteins

  • Potential for higher yields of soluble protein

  • More sophisticated post-translational processing

For the expression of functional ExoVII complex, co-expression strategies would be necessary:

• Bicistronic vectors containing both xseA and xseB genes

• Dual plasmid systems with compatible origins of replication

• Stoichiometrically balanced expression of both subunits

When expressing XseA alone, consideration should be given to its large size (~50-60 kDa) and complex domain structure, which might require specialized approaches such as:

• Lower induction temperatures (16-25°C)

• Addition of solubility-enhancing fusion partners (MBP, SUMO)

• Co-expression with molecular chaperones

How can the solubility and stability of recombinant B. henselae XseA be optimized during purification?

Optimizing the solubility and stability of recombinant B. henselae XseA during purification would likely require several strategies:

• Buffer optimization:

  • pH range screening (typically 7.0-8.5 for nucleases)

  • Salt concentration optimization (typically 100-500 mM NaCl)

  • Addition of stabilizing agents: glycerol (10-20%), reducing agents (DTT, β-mercaptoethanol)

  • Inclusion of protease inhibitors to prevent degradation

• Solubilization approaches:

  • If inclusion bodies form, mild solubilization protocols using non-denaturing detergents

  • On-column refolding during affinity purification

  • Gradual removal of solubilizing agents through dialysis

• Storage conditions:

  • Flash-freezing in liquid nitrogen with cryoprotectants

  • Addition of stabilizers like trehalose or sucrose

  • Optimized protein concentration to prevent aggregation

• Additional considerations for ExoVII complex:

  • Maintaining the appropriate XseA:XseB stoichiometry throughout purification

  • Using gentle purification methods to preserve complex integrity

  • Size-exclusion chromatography to isolate properly assembled complexes

For purification of the intact ExoVII complex, approaches similar to those used for structural studies of E. coli ExoVII could be adapted, which produced sufficient quantities of stable complex for cryoEM analysis .

What analytical methods are most effective for characterizing the purity, folding, and activity of recombinant B. henselae XseA?

A comprehensive characterization of recombinant B. henselae XseA would employ multiple analytical methods to assess purity, folding, and enzymatic activity:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining

  • Size-exclusion chromatography

  • Mass spectrometry to confirm identity and detect contaminants

  • Western blotting with specific antibodies, if available

• Structural integrity and folding:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Fluorescence spectroscopy to evaluate tertiary structure

  • Differential scanning fluorimetry (DSF) for thermal stability assessment

  • Limited proteolysis to probe domain organization

• Functional characterization:

  • Nuclease activity assays using labeled DNA substrates

  • Gel-shift assays to assess DNA binding capabilities, similar to those used for ExoI variants

  • Kinetic analyses to determine reaction rates and substrate preferences

  • Structure-function studies using site-directed mutagenesis

• Quaternary structure analysis:

  • Native PAGE to assess complex formation

  • Analytical ultracentrifugation to determine stoichiometry

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Negative-stain electron microscopy to visualize complex architecture

For the XseA-XseB complex, additional analyses would focus on subunit interactions and complex stability, including crosslinking studies and assembly kinetics. The combination of these analytical approaches would provide a comprehensive characterization of the recombinant protein and its enzymatic properties.