Recombinant Bradyrhizobium japonicum Holliday junction ATP-dependent DNA helicase RuvB (ruvB)

What is the fundamental role of RuvB helicase in Bradyrhizobium japonicum?

The RuvB protein in B. japonicum, like its homologs in other bacteria, functions as an ATP-dependent DNA helicase that promotes branch migration of Holliday junctions during homologous recombination and DNA repair processes. When working in concert with RuvA (which provides junction specificity), the RuvB ATPase supplies the motor force necessary for branch migration - a critical step in the formation of heteroduplex DNA and resolution of recombination intermediates .

Methodologically, the function of RuvB can be assessed through in vitro strand displacement assays using partially duplex DNA substrates. The polarity of this unwinding (5'→3' in E. coli) and its ATP dependence are defining characteristics that should be examined when studying the B. japonicum homolog .

How does RuvB from B. japonicum differ structurally from well-characterized homologs in other bacterial species?

While the search results don't specifically characterize the B. japonicum RuvB protein structure, comparative genomic approaches would be essential to identify unique structural features. Based on E. coli studies, RuvB functions as a hexameric ring-shaped motor protein that encircles DNA and uses ATP hydrolysis to drive branch migration .

To determine structural differences, researchers should:

• Perform sequence alignment of B. japonicum RuvB with homologs from E. coli and other bacteria

• Identify conserved domains and unique sequence features

• Generate structural predictions using homology modeling

• Validate through experimental approaches such as X-ray crystallography or cryo-EM

The high conservation of DNA repair mechanisms suggests functional similarity, but species-specific adaptations may exist that could influence protein-protein interactions or substrate specificity.

What expression systems are most effective for producing recombinant B. japonicum RuvB protein?

For effective recombinant expression of B. japonicum RuvB, researchers should consider:

• E. coli-based expression systems: BL21(DE3) strains commonly serve as effective hosts for bacterial recombinant proteins.

• Codon optimization: B. japonicum has different codon usage patterns than E. coli, so codon optimization may improve expression levels.

• Fusion tags: A hexahistidine tag facilitates purification via nickel affinity chromatography, while alternative tags like GST can improve solubility.

• Expression conditions: Lower temperatures (16-20°C) often improve protein folding and solubility, especially for ATPases.

The expression construct should include appropriate promoters (T7 is commonly used) and ribosome binding sites optimized for the host system. Purification typically involves multiple chromatography steps, including affinity, ion exchange, and size exclusion chromatography to ensure protein homogeneity for functional assays.

What are the basic assays to confirm ATP-dependent helicase activity of recombinant B. japonicum RuvB?

To confirm ATP-dependent helicase activity of purified recombinant B. japonicum RuvB, researchers should perform:

• ATP hydrolysis assays: Measuring inorganic phosphate release using colorimetric methods (malachite green) or radioactive [γ-32P]ATP to confirm ATPase activity.

• DNA unwinding assays: Using partially duplex DNA substrates (similar to those described for E. coli RuvB) with fluorescently labeled oligonucleotides to monitor strand displacement .

• Branch migration assays: Using synthetic Holliday junction structures to assess the ability of RuvB (with or without RuvA) to promote branch migration.

These assays should include appropriate controls:

• ATP vs. non-hydrolyzable ATP analogs

• RuvB alone vs. RuvB with RuvA

• Different DNA substrate lengths to assess efficiency relationships

Based on E. coli studies, the efficiency of DNA unwinding is expected to be inversely related to the length of duplex DNA, which should be verified for the B. japonicum protein .

How does the interaction between RuvA and RuvB in B. japonicum influence branch migration activity and how can these interactions be studied?

The interaction between RuvA and RuvB proteins is critical for efficient branch migration of Holliday junctions. In E. coli, RuvA binds specifically to Holliday junctions, reducing the requirement for RuvB by approximately 50-fold by providing target specificity .

To study this interaction in B. japonicum:

• Protein-protein interaction assays:

  • Pull-down assays using tagged RuvA to capture RuvB

  • Surface plasmon resonance to quantify binding kinetics

  • Isothermal titration calorimetry to determine thermodynamic parameters

• Functional reconstitution experiments:

  • Titration experiments varying RuvA:RuvB ratios to determine optimal stoichiometry

  • Assessing branch migration rates with and without RuvA

  • Testing high concentrations of RuvB alone to determine if it can overcome the requirement for RuvA as observed in E. coli systems

• Structural studies of the complex:

  • Cryo-EM of the RuvAB-Holliday junction complex

  • Cross-linking mass spectrometry to identify interaction interfaces

Understanding these interactions would provide insights into whether the B. japonicum RuvAB system has evolved unique regulatory mechanisms compared to model systems.

What is the impact of environmental stress conditions on the expression and activity of RuvB in B. japonicum?

Given that RuvB in E. coli is induced as part of the SOS response to DNA damage , examining stress responses in B. japonicum would be valuable research:

• Expression analysis under stress conditions:

  • Measure ruvB transcript levels using RT-qPCR following exposure to:

    • UV radiation

    • Oxidative stress (H₂O₂, paraquat)

    • Desiccation

    • pH stress

    • Symbiotic conditions (plant root exudates)

• Protein level assessment:

  • Western blotting with anti-RuvB antibodies

  • Proteomics analysis under various stress conditions

• Functional impact:

  • In vitro activity assays using protein extracts from stressed bacteria

  • Recombination frequency measurements under stress conditions

  • DNA damage repair kinetics following stress exposure

These studies would reveal whether B. japonicum modulates RuvB activity as part of its adaptation to the soil environment or symbiotic lifestyle, potentially revealing unique regulatory mechanisms compared to non-symbiotic bacteria.

How can CRISPR-Cas9 technology be applied to study the in vivo function of RuvB in B. japonicum?

CRISPR-Cas9 technology offers powerful approaches to study RuvB function in B. japonicum:

• Gene knockout strategies:

  • Design sgRNAs targeting the ruvB gene

  • Introduce repair templates containing antibiotic resistance markers

  • Screen for successful integration and gene disruption

  • Assess phenotypic consequences on:

    • Growth rate and cell morphology

    • Recombination frequency

    • DNA damage sensitivity

    • Symbiotic efficiency with host plants

• Gene editing for functional studies:

  • Create point mutations in ATPase domains to examine catalytic residues

  • Introduce fluorescent protein fusions for localization studies

  • Engineer epitope tags for immunoprecipitation experiments

• Inducible expression systems:

  • Replace native promoter with inducible systems

  • Study effects of RuvB overexpression or depletion

  • Temporal control of expression during symbiosis

When working with B. japonicum, electroporation of CRISPR-Cas9 components has been shown to be effective for genetic manipulation, though optimization of transformation protocols may be required for different strains .

What is the relationship between RuvB function and the symbiotic efficiency of B. japonicum?

Understanding the link between DNA repair mechanisms and symbiotic performance represents an important research direction:

• Comparative genomics and expression analysis:

  • Compare ruvB sequences and expression levels between strains with different symbiotic efficiencies

  • Analyze RNA-seq data from nodules at different developmental stages

  • Examine whether highly competitive strains (e.g., strain 5873) show differences in RuvB sequence or expression

• Genetic manipulation studies:

  • Create ruvB mutants with altered activity levels

  • Assess their competitive nodulation ability

  • Evaluate nitrogen fixation efficiency

  • Measure persistence in soil and rhizosphere

• Environmental stress connection:

  • Test whether strains with altered RuvB function show different responses to environmental stresses encountered during symbiosis

  • Examine whether DNA damage repair capacity correlates with symbiotic persistence

This table framework identifies key parameters to measure when examining the relationship between RuvB function and symbiotic performance.

How can high-throughput screening approaches be developed to identify small molecule modulators of B. japonicum RuvB activity?

Developing screens for RuvB modulators could provide valuable research tools:

• Assay development:

  • Adapt ATPase activity assays to microplate format using colorimetric phosphate detection

  • Develop FRET-based DNA unwinding assays using fluorescently labeled oligonucleotides

  • Optimize signal-to-noise ratios and Z' factors for screening applications

• Compound library screening:

  • Test focused libraries of known DNA-binding compounds and ATPase inhibitors

  • Screen natural product extracts, particularly from plant root exudates

  • Employ diversity-oriented synthetic libraries

• Hit validation and characterization:

  • Confirm activity with dose-response curves

  • Determine mechanism of action (competitive vs. non-competitive inhibition)

  • Assess selectivity against other DNA helicases

  • Evaluate effects on bacterial growth and symbiotic functions

• In vivo applications:

  • Test effects of validated compounds on:

    • DNA repair efficiency

    • Recombination frequency

    • Stress adaptation

    • Symbiotic performance

Such modulators could serve as valuable research tools for dissecting RuvB function in vivo and potentially lead to applications in agricultural biotechnology by modulating symbiotic efficiency.

What strategies can address poor solubility of recombinant B. japonicum RuvB protein?

Researchers frequently encounter solubility issues when expressing recombinant DNA helicases:

• Expression optimization approaches:

  • Lower induction temperatures (16-20°C)

  • Reduced inducer concentrations

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Alternative expression hosts (e.g., Arctic Express strains with cold-adapted chaperones)

• Protein engineering solutions:

  • Test different solubility-enhancing fusion tags (MBP, SUMO, GST)

  • Design truncated constructs based on domain prediction

  • Remove hydrophobic regions predicted to cause aggregation

  • Introduce surface mutations to enhance solubility

• Buffer optimization:

  • Screen additives (glycerol, arginine, detergents)

  • Test various salt concentrations and pH conditions

  • Include stabilizing cofactors (ATP, ADP, Mg²⁺)

  • Use deuterated solvents for structural studies

The hexameric nature of RuvB may complicate expression, so approaches that stabilize oligomeric states (such as chemical crosslinking or co-expression with RuvA) might enhance functional protein yields.

How can researchers address contaminating nuclease activity in purified B. japonicum RuvB preparations?

Nuclease contamination can confound helicase assays, requiring specific mitigation strategies:

• Purification optimizations:

  • Include EDTA in initial lysis buffers to inhibit nucleases

  • Add nuclease inhibitors (PMSF, benzamidine, RNasin)

  • Incorporate additional purification steps:

    • Heparin affinity chromatography

    • Hydroxyapatite chromatography

    • Size exclusion with stringent cutoffs

• Activity verification methods:

  • Controls with ATP vs. without ATP (true helicase activity is ATP-dependent)

  • DNA unwinding assays with and without RuvA to confirm specificity

  • Substrate specificity tests (Holliday junctions vs. random DNA)

  • Activity in the presence of nuclease inhibitors or EDTA

• Contaminant identification:

  • Mass spectrometry analysis of purified fractions

  • Activity-based protein profiling for nuclease detection

  • Antibody-based detection of common contaminants

Researchers should also consider designing nuclease-resistant DNA substrates with modified backbones for initial testing of potentially contaminated preparations.

What single-molecule approaches can be applied to study the mechanistic details of B. japonicum RuvB activity?

Single-molecule techniques offer unique insights into helicase mechanism:

• Single-molecule FRET (smFRET):

  • Design DNA substrates with strategically placed fluorophores

  • Monitor real-time conformational changes during branch migration

  • Determine step size and rate of RuvB-mediated unwinding

  • Analyze the effect of ATP concentration on unwinding kinetics

• Optical/magnetic tweezers:

  • Attach DNA substrates between beads/surfaces

  • Apply controlled force while measuring extension

  • Determine the force generation capacity of RuvB

  • Measure pausing, backtracking, and processivity

• DNA curtains and total internal reflection fluorescence (TIRF):

  • Visualize multiple DNA molecules in parallel

  • Track fluorescently labeled RuvB movement along DNA

  • Analyze the assembly of RuvAB complexes on Holliday junctions

  • Quantify residence times and processivity

These approaches would provide critical insights into whether B. japonicum RuvB displays distinct mechanistic properties compared to the well-characterized E. coli homolog, potentially revealing adaptations relevant to its symbiotic lifestyle.

How can structural studies of B. japonicum RuvB advance our understanding of its mechanism and species-specific functions?

Structural biology approaches offer powerful insights:

• X-ray crystallography strategies:

  • Crystallize RuvB in different nucleotide-bound states (apo, ATP, ADP)

  • Co-crystallize with RuvA and/or DNA substrates

  • Solve structures to identify catalytic residues and conformational changes

  • Compare with E. coli homologs to identify species-specific features

• Cryo-electron microscopy applications:

  • Visualize RuvB hexamers on DNA substrates

  • Capture different states of the ATP hydrolysis cycle

  • Reconstruct the complete RuvAB-Holliday junction complex

  • Determine high-resolution structures without crystallization

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map protein dynamics during ATP binding and hydrolysis

  • Identify regions involved in DNA interaction

  • Characterize conformational changes during branch migration

  • Compare with other bacterial RuvB proteins

• Integration with functional studies:

  • Structure-guided mutagenesis to validate catalytic mechanisms

  • Design of B. japonicum-specific inhibitors or activity modulators

  • Engineering of RuvB variants with enhanced activity or altered specificity

These structural studies could potentially reveal adaptations that might be relevant to B. japonicum's function in the soil environment or during symbiotic interactions.

What is the potential role of RuvB in horizontal gene transfer and genomic adaptation in Bradyrhizobium species?

Given RuvB's role in recombination, its potential involvement in horizontal gene transfer merits investigation:

• Comparative genomic approaches:

  • Analyze genomic islands in B. japonicum strains with different RuvB variants

  • Assess correlation between RuvB sequence/expression and genomic plasticity

  • Compare recombination hotspots across Bradyrhizobium species

• Experimental evolution studies:

  • Subject wild-type and RuvB-modified strains to selective pressures

  • Measure rates of adaptive gene acquisition

  • Track genomic changes during host adaptation

• Horizontal gene transfer assays:

  • Develop laboratory systems to measure conjugation efficiency

  • Test integration rates of foreign DNA

  • Assess recombination frequency between genomic regions

This research direction could reveal whether RuvB function contributes to the remarkable genomic diversity observed among Bradyrhizobium strains and their adaptation to different host plants and environmental conditions .

How might RuvB function contribute to the competitive nodulation ability of elite B. japonicum strains?

The competitive advantage of certain B. japonicum strains in nodulation might relate to DNA repair capabilities:

• Competitive nodulation assays:

  • Compare wild-type and RuvB-modified strains in mixed inoculation experiments

  • Use strain-specific PCR assays (similar to those developed for strain 5873) to track nodule occupancy

  • Analyze competitive index under various environmental stress conditions

• Stress response characterization:

  • Assess DNA damage accumulation during rhizosphere colonization

  • Measure repair efficiency following exposure to plant defense responses

  • Correlate DNA repair capacity with competitive fitness

• Field trials with modified strains:

  • Test performance of strains with altered RuvB function

  • Assess persistence in agricultural soils over multiple growing seasons

  • Measure nitrogen fixation efficiency in competitive environments

This research could provide insights into whether DNA repair and recombination functions contribute to the field performance of commercial inoculant strains, potentially leading to improved strain selection criteria or engineering approaches.

Could RuvB function be targeted to improve agricultural applications of B. japonicum?

Potential agricultural applications warrant exploration:

These applications could contribute to more resilient and effective nitrogen-fixing inoculants, reducing the need for chemical fertilizers in soybean and other legume crops.

What are the optimal experimental controls when studying recombinant B. japonicum RuvB activity?

Rigorous experimental design requires appropriate controls:

• Negative controls:

  • ATPase-deficient RuvB mutants (Walker A/B mutations)

  • Reactions without ATP or with non-hydrolyzable ATP analogs

  • Heat-inactivated protein preparations

  • Non-specific DNA substrates lacking Holliday junctions

• Positive controls:

  • Well-characterized E. coli RuvB as reference standard

  • Commercial helicases with known activity profiles

  • RuvA-RuvB combinations known to be active

• Specificity controls:

  • Competition experiments with unlabeled substrates

  • Inhibitor studies (e.g., ATPase inhibitors)

  • RuvB from related Bradyrhizobium species

• System validation:

  • Demonstrate ATP concentration dependence

  • Show expected DNA substrate length effects

  • Confirm RuvA enhancement of activity

  • Verify temperature and buffer condition optima

These controls ensure that observed activities are specifically attributable to B. japonicum RuvB rather than contaminants or experimental artifacts.

What bioinformatic approaches are most valuable for analyzing RuvB conservation and evolution across Bradyrhizobium species?

Computational analyses provide important evolutionary insights:

• Sequence analysis methods:

  • Multiple sequence alignment of RuvB proteins across Bradyrhizobium species

  • Phylogenetic tree construction using maximum likelihood methods

  • Identification of conserved domains and catalytic motifs

  • Detection of positive selection signatures using dN/dS analysis

• Structural bioinformatics:

  • Homology modeling based on E. coli RuvB crystal structures

  • Molecular dynamics simulations to compare conformational dynamics

  • Protein-protein interaction interface prediction for RuvA-RuvB complex

  • Virtual screening for potential species-specific inhibitors

• Genomic context analysis:

  • Examination of ruvB gene neighborhood across species

  • Identification of co-evolving gene partners

  • Analysis of regulatory elements and promoter regions

  • Correlation of genetic variations with symbiotic host range