Recombinant Tropheryma whipplei Holliday junction ATP-dependent DNA helicase RuvB (ruvB)

What is the fundamental role of RuvB in bacterial DNA recombination?

RuvB functions as an ATP-dependent DNA helicase that works in conjunction with RuvA to promote branch migration of Holliday junctions. Based on studies in Escherichia coli, RuvA specifically interacts with Holliday junctions while RuvB provides the motor function through its ATPase activity . Together, they promote the movement of the junction along DNA, a process critical for forming heteroduplex DNA during genetic recombination . The RuvA and RuvB proteins also demonstrate DNA helicase activity, unwinding partially duplex DNA with a 5'→3' polarity in an ATP-dependent manner . This mechanism is essential for normal levels of genetic recombination and DNA repair in bacteria.

What genomic context surrounds the ruvB gene in T. whipplei?

The T. whipplei genome exhibits no detectable colinearity with its close relatives that have much larger genomes, such as Mycobacterium leprae (1605 ORFs), Corynebacterium glutamicum (3040 ORFs), and Mycobacterium tuberculosis (3927 ORFs) . The genomic context of ruvB in T. whipplei is likely unique due to the extensive genome rearrangements observed in this organism. These rearrangements are frequently triggered by repeats within paralogous genes, particularly those encoding cell-surface proteins . T. whipplei contains site-specific integrase/recombinase genes (xerC and xerD) that may be involved in these genome rearrangements , potentially affecting the genomic neighborhood of ruvB.

What are the optimal conditions for recombinant expression of T. whipplei RuvB?

Based on T. whipplei's unique genomic characteristics, recombinant expression should account for the organism's unusual features. T. whipplei has deficiencies in amino acid metabolisms , suggesting that expression systems should be supplemented with appropriate amino acids. When designing an expression system:

• Host selection: E. coli BL21(DE3) strains are recommended due to their reduced protease activity and compatibility with T7 promoter-based expression systems.

• Codon optimization: Since T. whipplei has a 46% G+C content , which is unusually low for Actinobacteria, codon optimization may improve expression efficiency in standard hosts.

• Expression conditions: Initial expression trials should test multiple conditions:

  • Temperature: 16°C, 25°C, and 37°C

  • IPTG concentration: 0.1 mM to 1.0 mM

  • Expression time: 4 hours to overnight

• Solubility enhancement: Consider fusion partners (MBP, SUMO, or GST) to improve solubility of the recombinant protein.

What purification strategy is most effective for isolating active T. whipplei RuvB protein?

A multi-step purification approach is recommended for obtaining highly pure and active RuvB protein:

• Initial capture: Affinity chromatography using either:

  • Ni-NTA (for His-tagged protein)

  • Amylose resin (for MBP fusion)

  • Glutathione sepharose (for GST fusion)

• Intermediate purification: Ion exchange chromatography

  • Based on the theoretical pI of T. whipplei RuvB

• Polishing step: Size exclusion chromatography

  • To separate monomeric from oligomeric forms and remove aggregates

• Buffer optimization: Since RuvB functions as an ATPase, the purification and storage buffers should include:

  • 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0)

  • 100-300 mM NaCl

  • 1-5 mM MgCl₂ (for stabilizing the ATPase domain)

  • 1-5 mM DTT or 0.5-2 mM TCEP (to maintain reduced cysteines)

  • 10% glycerol (for storage stability)

• Activity preservation: Add ADP or non-hydrolyzable ATP analog (e.g., AMP-PNP) at 0.1-0.5 mM to stabilize the protein during purification and storage.

How can the ATP-dependent helicase activity of T. whipplei RuvB be assayed reliably?

Several complementary approaches can be used to assess the helicase activity:

• Strand displacement assay:

  • Construct partially duplex DNA substrates with 5'-labeled oligonucleotides (32P or fluorescent labels)

  • Incubate with purified RuvB (with and without RuvA) in the presence of ATP

  • Analyze products by native PAGE to quantify strand displacement

  • Controls should include ATP-absent reactions and heat-denatured substrates

• Real-time assays:

  • FRET-based methods using dual-labeled substrates

  • Stop-flow techniques to measure reaction kinetics

• ATPase activity measurement:

  • Malachite green assay for phosphate release

  • Coupled enzyme assay with pyruvate kinase and lactate dehydrogenase

What functional domains of T. whipplei RuvB are critical for its interaction with RuvA?

Based on studies of RuvB in other bacteria, several domains would be critical for proper RuvB function:

• N-terminal domain: Contains the nucleotide-binding pocket (Walker A motif) essential for ATP binding.

• Central domain: Houses the Walker B motif involved in ATP hydrolysis and likely contains residues for RuvA interaction.

• C-terminal domain: Often involved in oligomerization and potentially in DNA interaction.

Experimental approaches to identify critical interaction domains include:

• Site-directed mutagenesis of conserved residues

• Truncation analysis to determine minimal functional domains

• Cross-linking coupled with mass spectrometry to map interaction surfaces

• Yeast two-hybrid or bacterial two-hybrid assays to assess protein-protein interactions

• Hydrogen-deuterium exchange mass spectrometry to identify interaction surfaces

What is the ATP hydrolysis mechanism of T. whipplei RuvB and how does it drive branch migration?

The ATP hydrolysis mechanism likely follows a conserved pattern seen in other AAA+ ATPases:

• Sequential hydrolysis model: ATP hydrolysis occurs in a sequential manner around the hexameric ring.

• Allosteric regulation: Binding of ATP to one subunit affects the conformation and activity of adjacent subunits.

• Mechanical coupling: ATP hydrolysis causes conformational changes that are transmitted to the DNA substrate, resulting in translocation along the DNA strand.

• Coordination with RuvA: The RuvA tetramer maintains the Holliday junction in an open, square-planar configuration, while RuvB hexamers on opposing arms drive branch migration through coordinated ATP hydrolysis.

To study this mechanism specifically for T. whipplei RuvB, researchers should consider:

• Pre-steady-state kinetic analysis of ATP hydrolysis

• Structure determination of different nucleotide-bound states

• Single-molecule approaches to directly observe branch migration events

• FRET-based assays to detect conformational changes during the reaction cycle

How can studies of T. whipplei RuvB contribute to understanding Whipple's disease pathogenesis?

T. whipplei RuvB studies may provide valuable insights into Whipple's disease pathogenesis in several ways:

• DNA repair and persistence: RuvB's role in DNA repair may contribute to T. whipplei's ability to persist in host tissues. Whipple's disease is a chronic condition, and the bacterium's DNA repair mechanisms might play a role in its long-term survival .

• Genomic plasticity: T. whipplei exhibits genome rearrangements that may be influenced by recombination processes involving RuvB. These rearrangements are associated with a large cell-surface protein family and might represent a mechanism for evading host defenses .

• Antibiotic resistance: T. whipplei has a mutation in DNA gyrase predicting resistance to quinolone antibiotics . Understanding how RuvB interacts with other DNA repair pathways might provide insights into antibiotic resistance mechanisms.

• Adaptation to host environment: T. whipplei's reduced genome reflects adaptation to its human host niche . Studying how essential processes like DNA repair are maintained in this reduced genome context could reveal adaptations crucial for pathogenesis.

What are the methodological challenges in studying T. whipplei RuvB in the context of infection models?

Researchers face several key challenges when studying T. whipplei RuvB in infection models:

Potential approaches to address these challenges include:

• Developing improved culture conditions based on T. whipplei's metabolic requirements

• Using heterologous expression systems to study RuvB function

• Employing cell culture infection models with human macrophages or intestinal cell lines

• Developing conditional expression systems for essential genes like ruvB

How does T. whipplei's reduced genome influence the functional importance of RuvB?

The reduced genome of T. whipplei (927,303 bp encoding 808 predicted protein-coding genes) likely affects the functional importance of RuvB in several ways:

• Increased reliance on core processes: With fewer redundant pathways, T. whipplei may rely more heavily on essential processes like RuvB-mediated DNA repair.

• Conservation of function: Despite genome reduction, T. whipplei has retained genes for site-specific recombination (xerC and xerD) , suggesting the importance of DNA recombination processes.

• Integration with limited DNA repair capacity: T. whipplei shows deficiencies in various metabolic pathways . Its DNA repair repertoire is likely similarly reduced, potentially making the RuvAB pathway even more critical.

• Specialized adaptation: The retained functions of RuvB might be specifically adapted to T. whipplei's lifestyle as an intracellular pathogen, possibly focusing on repair of specific types of DNA damage encountered during infection.

What new experimental approaches could advance our understanding of T. whipplei RuvB function?

Several cutting-edge approaches could significantly advance our understanding:

• CRISPR interference (CRISPRi): For conditional knockdown of ruvB expression in T. whipplei to assess its essentiality and functional effects.

• Single-molecule techniques:

  • Optical tweezers to directly measure the force generated during branch migration

  • Single-molecule FRET to observe conformational changes during RuvB action

  • DNA curtains to visualize multiple RuvB complexes acting on DNA substrates

• Advanced structural approaches:

  • Cryo-EM of RuvB-RuvA-Holliday junction complexes

  • Integrative structural biology combining X-ray crystallography, NMR, and computational modeling

  • Time-resolved structural studies to capture different states of the reaction cycle

• Systems biology approaches:

  • Interaction networks to identify partners of RuvB beyond RuvA

  • Transcriptomics to identify conditions that regulate ruvB expression

  • Comparative genomics across clinical isolates to identify variations in the ruvB gene

• Synthetic biology approaches:

  • Reconstitution of minimal DNA repair systems incorporating T. whipplei RuvB

  • Creating chimeric proteins to identify species-specific functional domains

How might the study of T. whipplei RuvB inform the development of new diagnostic tools for Whipple's disease?

Understanding T. whipplei RuvB could contribute to improved diagnostics in several ways:

• Molecular detection: Knowledge of ruvB sequence conservation across T. whipplei strains could inform the development of PCR primers for specific detection, complementing existing molecular tests .

• Protein-based diagnostics: If RuvB contains T. whipplei-specific epitopes, antibodies against these regions could be developed for immunodiagnostic tests.

• Functional biomarkers: Understanding RuvB's role in DNA repair might reveal specific DNA repair signatures or metabolites that could serve as biomarkers for active infection.

• Distinguishing active from persistent infection: If RuvB activity correlates with bacterial replication, assays targeting this activity might help distinguish active disease from persistent infection.

• Predicting treatment response: Knowledge of RuvB's contribution to bacterial persistence might help predict treatment outcomes and inform therapeutic decisions.

What data contradictions exist in the current literature regarding bacterial RuvB proteins, and how might these be resolved through studies of T. whipplei RuvB?

Several unresolved questions and contradictions exist in the literature regarding bacterial RuvB proteins:

• Helicase directionality: Some studies indicate 5'→3' polarity for RuvAB-mediated branch migration , while others suggest a different mechanism. T. whipplei RuvB could be studied to resolve this contradiction.

• ATP hydrolysis coupling: The precise mechanism coupling ATP hydrolysis to DNA movement remains debated. Structural studies of T. whipplei RuvB in different nucleotide-bound states could provide insights.

• RuvB oligomerization state: While typically described as a hexamer, some studies suggest alternative oligomeric forms under certain conditions. Analytical ultracentrifugation and native mass spectrometry of T. whipplei RuvB could address this question.

• Interaction with other repair proteins: The extent of functional overlap and interaction between the RuvABC pathway and other DNA repair pathways varies across bacterial species. Studies in T. whipplei, with its reduced genome, might clarify these relationships.

• Evolutionary adaptation: How RuvB function has adapted to different bacterial lifestyles remains poorly understood. The study of T. whipplei RuvB, from a host-adapted pathogen with a reduced genome, could provide valuable evolutionary insights.