Recombinant Bradyrhizobium japonicum Recombination protein RecR (recR)

What is the functional role of RecR protein in Bradyrhizobium japonicum?

RecR in Bradyrhizobium japonicum functions as a critical component in homologous recombination and DNA repair pathways. It forms part of the RecFOR pathway that assists RecA protein in DNA strand exchange during recombinational repair. Specifically, RecR works in concert with RecF and RecO to facilitate RecA loading onto single-stranded DNA (ssDNA) that is coated with single-stranded DNA binding (Ssb) protein. This process is particularly important in the repair of DNA gaps and in overcoming the inhibitory effect of Ssb protein on RecA nucleoprotein filament formation.

Experimental evidence indicates that RecR in bacterial systems forms a heterotrimeric complex with RecF and RecO in a 1:1:1 molar ratio, with a molecular weight of approximately 276 kDa, suggesting a multimeric arrangement of these proteins during their function . This complex serves as an "anti-Ssb factor," essential for protecting the genome integrity of slow-growing organisms like B. japonicum during their extended growth cycles.

How does RecR interact with RecF and RecO to facilitate recombination in B. japonicum?

The interaction between RecR, RecF, and RecO in B. japonicum is sequential and highly regulated:

• RecF initially interacts with RecO through direct protein-protein contacts

• RecR then interacts with the RecF-RecO complex to form the complete RecF-RecO-RecR heterotrimer

• RecO mediates the interactions between RecF and RecR, acting as a bridge between these proteins

• The formed complex can then interact with Ssb-coated ssDNA

Immunoprecipitation experiments have demonstrated that RecR is precipitated by anti-RecF antibodies only when both RecO and RecF are present, indicating that RecO mediates the RecF-RecR interaction . This mediation is crucial for B. japonicum, which relies heavily on efficient DNA repair mechanisms due to its slow growth rate and susceptibility to DNA damage during symbiotic nitrogen fixation processes.

The presence of ATP can modulate these interactions, with ATP binding to RecF decreasing its affinity for RecO without affecting RecO's affinity for Ssb. This ATP-dependent regulation allows for fine-tuning of the recombination process in response to cellular energy states .

How does the genetic organization of the recR locus in B. japonicum compare to other bacteria?

The recR gene in B. japonicum is located on its single circular chromosome, which is 9,105,828 bp in length with an average GC content of 64.1% . While the complete genomic context of recR in B. japonicum has not been fully characterized in the provided search results, general bacterial organizational patterns suggest it may be part of a conserved genomic arrangement.

In bacterial systems, recR is often found in operons with other DNA repair genes, although the specific arrangement can vary between species. The genetic organization comparison between B. japonicum and other nitrogen-fixing bacteria such as B. diazoefficiens USDA110 would be particularly informative, as B. japonicum strains have been reclassified into different species based on genomic analyses .

A comparative analysis table of recR gene location and organization would show:

Further genomic analyses are required to fully map the recR locus in different Bradyrhizobium strains and species.

How does the RecFOR pathway in B. japonicum contribute to its symbiotic relationship with legumes?

The RecFOR pathway, including RecR, plays a crucial role in maintaining genomic integrity during the transition from free-living to symbiotic states in B. japonicum. During nodule formation and nitrogen fixation in soybean roots, B. japonicum faces various stressors including oxidative stress and host defense responses that can damage DNA.

Methodological approaches to study this contribution include:

• Creation of recR knockout mutants in B. japonicum to assess:

  • Nodulation efficiency with soybean plants

  • Nitrogen fixation capacity (acetylene reduction assay)

  • Survival rates under oxidative stress conditions

• Comparative transcriptomic analysis of recR expression between:

  • Free-living bacteria

  • Bacteria during infection thread formation

  • Bacteroids within mature nodules

Research suggests that efficient DNA repair systems are particularly important for B. japonicum due to its slow growth characteristics (doubling time significantly longer than other bacteria) . The recombination proteins including RecR likely contribute to the bacterium's ability to maintain genomic stability during the dramatic physiological changes that occur during symbiosis establishment.

The ability of B. japonicum to form successful root nodules depends on proper functioning of DNA repair pathways, as genomic instability could compromise the complex signaling between plant and bacterium during symbiotic establishment.

What methods are most effective for expressing and purifying recombinant RecR from B. japonicum?

Expressing and purifying recombinant RecR from B. japonicum presents several challenges due to the slow growth of the organism and potential solubility issues. Based on protocols for similar bacterial recombination proteins, the following methodological approach is recommended:

Expression System Selection:

• E. coli BL21(DE3) remains the system of choice, using vectors with tightly controlled promoters (pET series)

• Consider codon optimization for B. japonicum genes which have a high GC content (64.1%)

Optimized Protocol:

• Clone the recR gene from B. japonicum genomic DNA using PCR with high-fidelity polymerase

• Insert into pET-28a(+) vector with N-terminal His6-tag for purification

• Transform into E. coli BL21(DE3)

• Culture conditions:

  • Grow at 37°C until OD600 reaches 0.6-0.8

  • Induce with 0.1-0.5 mM IPTG

  • Shift to lower temperature (16-20°C) for overnight expression to improve solubility

Purification Strategy:

• Cell lysis using sonication in buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

• Ni-NTA affinity chromatography

• Size exclusion chromatography to ensure homogeneity

• Storage in buffer with 20-50% glycerol at -80°C

Protein quality assessment should include SDS-PAGE (>85% purity), western blot confirmation, and functional assays testing DNA binding and RecF-RecO interactions. The purified protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for stability during storage .

What techniques can be used to study the interactions between RecR and other recombination proteins in B. japonicum?

Several complementary techniques can be employed to characterize the interactions between RecR and other recombination proteins in B. japonicum:

In Vitro Techniques:

• Co-immunoprecipitation (Co-IP):

  • Use anti-RecR antibodies to precipitate protein complexes

  • Western blot analysis to identify interacting partners (RecF, RecO)

  • This approach confirmed the 1:1:1 stoichiometry of RecF-RecO-RecR complex in other bacteria

• Size-exclusion chromatography:

  • Analyze complex formation by monitoring elution profiles

  • Helps determine the molecular weight of protein complexes (RecF-RecO-RecR complex is approximately 276 kDa)

• Surface Plasmon Resonance (SPR):

  • Measure binding kinetics and affinity constants between RecR and potential partners

  • Immobilize RecR on sensor chip and flow other proteins to detect interactions

• Affinity chromatography:

  • Use Ssb protein affinity columns to detect formation of RecF-RecO-RecR-Ssb complexes

  • Analyze elution patterns at different salt concentrations to assess binding strength

In Vivo Techniques:

• Bacterial two-hybrid system:

  • Detect protein-protein interactions in living bacterial cells

  • Particularly useful for membrane or insoluble proteins

• Luciferase complementation imaging (LCI):

  • Similar to the technique used to study protein interactions in B. diazoefficiens-soybean symbiosis

  • Has successfully verified interactions between symbiotic proteins

• Fluorescence microscopy with fusion proteins:

  • Create RecR-GFP fusions to visualize localization in live cells

  • Co-localization studies with other fluorescently tagged recombination proteins

These methodologies should be adapted to account for the slow growth characteristics of B. japonicum, potentially requiring longer incubation times and specialized growth media such as BJSM (Bradyrhizobium japonicum selective medium) .

What are the most effective methods for creating recR mutants in B. japonicum?

Creating recR mutants in B. japonicum requires specialized approaches due to the organism's slow growth and high rates of spontaneous antibiotic resistance. Based on successful mutagenesis strategies in Bradyrhizobium, the following methodological approach is recommended:

Optimized Protocol for recR Mutagenesis:

• Construct preparation:

  • Design a construct where a kanamycin (Km) or spectinomycin (Sp) cassette replaces part of the recR gene

  • Include 1-2 kb of homologous flanking DNA sequences on each side of the antibiotic cassette

  • Clone this construct into a suicide vector that cannot replicate in B. japonicum

• Transformation method:

  • Electroporation of B. japonicum competent cells

  • Alternatively, triparental mating using E. coli donor and helper strains

• Selection protocol (addressing the high spontaneous resistance issue):

  • Use the rapid selection method developed for B. japonicum :
    a) Simple plate selection for antibiotic-resistant mutants
    b) Colony streaking for single colonies
    c) Direct colony lysis on nitrocellulose filters
    d) DNA hybridization to identify true recombinants without isolating genomic DNA

• Verification strategies:

  • PCR analysis with primers flanking the insertion site

  • Southern blot analysis to confirm single insertion event

  • Reverse transcription-PCR to verify absence of recR transcript

  • Western blot to confirm absence of RecR protein

This approach has been shown to increase efficiency in identifying recombinant site-directed mutants in B. japonicum by eliminating the need to first isolate genomic DNA from each potential mutant for Southern hybridization .

How can the phenotypic effects of recR mutations be assessed in B. japonicum?

The phenotypic effects of recR mutations in B. japonicum can be comprehensively assessed using a multi-faceted approach targeting both free-living and symbiotic states:

Free-living Growth Analysis:

• Growth curve determination:

  • Measure growth rates in rich and minimal media

  • Compare generation times between wild-type and recR mutants

  • Particularly important given B. japonicum's already slow growth characteristics

• Stress response profiling:

  • DNA damaging agents (UV radiation, mitomycin C, methyl methanesulfonate)

  • Oxidative stress inducers (H₂O₂, paraquat)

  • Measure survival rates and recovery times

  • This approach reveals RecR's role in DNA damage repair

• Microscopic examination:

  • Cell morphology assessment using phase contrast microscopy

  • Nucleoid structure analysis with DAPI staining

  • Potential filamentous growth indicating impaired DNA repair

Symbiotic Function Assessment:

• Nodulation assays with soybean plants:

  • Count nodule number per plant

  • Measure nodule size and morphology

  • Assess time to nodule appearance

  • Analyze bacteroid differentiation within nodules

• Nitrogen fixation quantification:

  • Acetylene reduction assay to measure nitrogenase activity

  • Plant dry weight and nitrogen content determination

  • Comparative assessment of plant height and leaf coloration

• Competitive nodulation experiments:

  • Co-inoculate wild-type and recR mutant strains

  • Use differential antibiotic resistance markers

  • Determine ratio of nodule occupancy

  • This would reveal if RecR function affects competitiveness

• Molecular analysis of symbiotic gene expression:

  • qRT-PCR for key symbiotic genes (nod, nif) in the mutant

  • RNA-seq to assess global transcriptional changes

  • This would reveal potential regulatory roles of RecR beyond DNA repair

The interconnection between DNA repair systems and symbiotic efficiency has been observed in other nitrogen-fixing bacteria, making these phenotypic analyses particularly relevant for understanding RecR function in B. japonicum.

What genomic approaches can reveal the evolution of RecR in Bradyrhizobium species?

Several genomic approaches can elucidate the evolutionary history of RecR across Bradyrhizobium species:

Multilocus Sequence Analysis (MLSA):

• Include recR as one of the protein-coding genes in MLSA along with established markers such as atpD, recA, glnII, and rpoB

• Compare evolutionary rates of recR with housekeeping genes across Bradyrhizobium species

• This approach has already proven effective for Bradyrhizobium taxonomy and biogeographic studies

Comparative Genomics:

• Synteny analysis:

  • Compare the genomic context of recR across multiple Bradyrhizobium genomes

  • Identify conservation or rearrangements in the recR locus

  • This could reveal functional constraints or mobile genetic element influence

• Selection pressure analysis:

  • Calculate dN/dS ratios to determine if recR is under purifying, neutral, or positive selection

  • Compare with other recombination genes (recF, recO, recA)

  • According to studies on B. japonicum populations, many genes show neutral equilibrium patterns

Detailed Population Genetic Metrics:
Based on methods used in Bradyrhizobium population studies , the following metrics should be calculated:

Phylogenomic Analysis:
Construct phylogenetic trees using:

• recR sequences alone

• Concatenated core genome sequences

• Compare topologies to identify potential horizontal gene transfer events

The high GC content (64.1%) of B. japonicum genome and the presence of numerous insertion sequences suggest genome plasticity that could affect recR evolution, particularly in comparison between B. japonicum and the reclassified B. diazoefficiens strains .

How can DNA binding properties of recombinant B. japonicum RecR be characterized?

The DNA binding properties of recombinant B. japonicum RecR can be characterized using multiple complementary biochemical and biophysical approaches:

Electrophoretic Mobility Shift Assay (EMSA):

• Prepare labeled DNA substrates that mimic recombination intermediates:

  • Single-stranded DNA (ssDNA)

  • Double-stranded DNA (dsDNA)

  • Gapped DNA structures

  • Holliday junctions

• Incubate with increasing concentrations of purified RecR

• Analyze mobility shifts on native polyacrylamide gels

• Include competition assays with unlabeled DNA to determine specificity

DNA Binding Kinetics:

• Surface Plasmon Resonance (SPR):

  • Immobilize DNA substrates on sensor chips

  • Flow RecR protein at various concentrations

  • Derive association (ka) and dissociation (kd) rate constants

  • Calculate equilibrium dissociation constant (KD)

• Fluorescence Anisotropy:

  • Use fluorescently labeled DNA oligonucleotides

  • Measure changes in anisotropy upon RecR binding

  • Determine binding affinities under various conditions

  • Test effect of Mg²⁺, ATP, and salt concentration on binding

Structural Studies:

• Atomic Force Microscopy (AFM):

  • Visualize RecR-DNA complexes at single-molecule resolution

  • Determine DNA conformation changes upon RecR binding

  • Assess potential DNA bridging or looping activities

• Electron Microscopy:

  • Negative staining to visualize RecR-DNA complexes

  • Analyze structural arrangements on DNA templates

Functional Assays:

• DNA Protection Assays:

  • Test if RecR protects DNA from nuclease digestion

  • Use DNase footprinting to identify specific binding sites

• ATP Hydrolysis Assays:

  • Determine if DNA binding stimulates any latent ATPase activity

  • Compare with RecF-RecO-RecR complex activity

• RecA Loading Assays:

  • Test the ability of RecR (alone or with RecF and RecO) to facilitate RecA loading onto SSB-coated ssDNA

  • This directly relates to the proposed function in presynaptic filament formation

Comparative Analysis:
Since the RecF-RecO-RecR complex functions as a heterotrimer , perform parallel assays with:

• RecR alone

• RecR + RecF

• RecR + RecO

• RecR + RecF + RecO
  to elucidate the contribution of RecR to the complex's DNA binding properties.

What approaches can determine the structural features of B. japonicum RecR protein?

Determining the structural features of B. japonicum RecR requires a multi-technique approach:

X-ray Crystallography:

• Crystallization screening:

  • Use vapor diffusion methods (hanging/sitting drop)

  • Screen commercial crystallization kits (Hampton Research, Molecular Dimensions)

  • Optimize promising conditions varying pH, salt, precipitant

  • Consider crystallization with DNA and/or RecF/RecO partners

• Data collection and processing:

  • Collect diffraction data at synchrotron facilities

  • Process data using standard crystallographic software (XDS, CCP4, PHENIX)

  • Solve structure by molecular replacement using known RecR structures

  • Build and refine model to obtain atomic resolution structure

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Express ¹⁵N and ¹³C labeled RecR in minimal media

• Collect multidimensional NMR spectra to assign backbone and side-chain resonances

• Determine secondary structure elements from chemical shift data

• Study dynamics and flexibility of specific domains

• Particularly useful for examining RecR in complex with RecF and RecO

Cryo-Electron Microscopy (Cryo-EM):

• Especially valuable for larger complexes (RecF-RecO-RecR, ~276 kDa)

• Prepare grids with RecR alone and in complex with partners

• Collect and process images using single-particle analysis

• Generate 3D reconstructions to visualize quaternary structure

Small-Angle X-ray Scattering (SAXS):

• Collect scattering data on RecR in solution

• Generate low-resolution molecular envelopes

• Particularly useful for flexible proteins or those difficult to crystallize

• Can provide insights into conformational changes upon complex formation

Computational Approaches:

• Homology modeling:

  • Use known RecR structures as templates

  • Validate models through molecular dynamics simulations

  • Predict key functional residues and domains

• AlphaFold2 or RoseTTAFold prediction:

  • Generate AI-based structural predictions

  • Compare with experimental data when available

  • Particularly useful for initial structural insights

Functional Domain Mapping:

• Limited proteolysis to identify domain boundaries

• Create deletion constructs to test domain functions

• Site-directed mutagenesis of predicted functional residues

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

These approaches would reveal important structural features of RecR, such as its DNA-binding domain, interaction surfaces with RecF and RecO, and any structural changes that occur upon complex formation or ATP binding.

How does the RecFOR pathway in B. japonicum compare to other DNA repair pathways in rhizobia?

The RecFOR pathway in B. japonicum represents one of several DNA repair mechanisms in rhizobia, each with distinct characteristics and evolutionary significance:

Comparative Analysis of DNA Repair Pathways in Rhizobia:

Methodological Approaches for Comparative Analysis:

• Genomic comparisons:

  • Identify homologs across Bradyrhizobium, Sinorhizobium, Rhizobium, and Mesorhizobium

  • Assess gene synteny and operon organization

  • Identify species-specific adaptations in RecFOR pathway components

• Functional complementation studies:

  • Express B. japonicum recR in E. coli recR mutants

  • Test if B. japonicum RecR can substitute for RecR in other rhizobia

  • Evaluate cross-species functionality of RecFOR complex formation

• Transcriptional regulation analysis:

  • Compare expression patterns of recF, recO, and recR genes

  • Identify regulatory elements (promoters, binding sites)

  • Assess coordination with nitrogen fixation genes

Evolutionary Considerations:
B. japonicum's slow growth rate (compared to other rhizobia) may have influenced the evolution of its DNA repair pathways. The RecFOR pathway is particularly important in slow-growing organisms where efficiently repairing DNA gaps is critical for genome stability during extended replication cycles.

The genome size difference between B. japonicum (9.1 Mb) and other rhizobia (typically 6-8 Mb) may also reflect different selective pressures on DNA repair systems, with larger genomes potentially requiring more robust repair mechanisms to maintain integrity.

Functional Specialization:
In B. japonicum, the RecFOR pathway likely plays an enhanced role in:

• Maintaining genomic stability during the bacteroid differentiation process

• Protecting DNA during oxidative stress encountered in nodules

• Repairing damage that occurs during the extended periods of nitrogen fixation

These specialized functions may be reflected in sequence adaptations or regulatory patterns unique to B. japonicum compared to other rhizobia.

How might RecR function be leveraged to improve agricultural applications of B. japonicum?

RecR function could be strategically leveraged to enhance B. japonicum's agricultural applications through several biotechnological approaches:

Enhanced Stress Tolerance:

• Controlled overexpression of recR:

  • Create strains with modestly increased RecR levels

  • Test survival under field-relevant stresses (heat, desiccation, UV)

  • The slow growth of B. japonicum and sensitivity to environmental stressors limit inoculant shelf-life

  • Enhanced DNA repair capacity could improve survival on seeds and in soil

• Development of stress-inducible expression systems:

  • Engineer recR expression to increase specifically during stress

  • Design synthetic promoters responsive to soil conditions

  • This approach could ensure optimal RecR levels exactly when needed

Improved Inoculant Formulations:

• Protective additives targeted to DNA repair function:

  • Include osmoprotective compounds that support RecR function

  • Optimize formulations based on RecR activity measurements

  • Current research shows osmoprotective compounds increase survival factors of B. japonicum on seeds

• Stability testing protocols:

  • Develop assays based on recR expression as biomarkers for inoculant viability

  • Use quantitative recovery factors to predict field performance

  • This addresses the difficulty in evaluating bacterial numbers on seeds

Enhanced Competitive Ability:

• Selective modification of RecR function:

  • Identify RecR variations associated with highly competitive strains

  • Introduce these variations into high nitrogen-fixing strains

  • This approach addresses the observed variation in competitiveness among B. japonicum strains

• Co-inoculation strategies:

  • Develop bacterial consortia where partner species protect B. japonicum DNA

  • Example: co-inoculation with Bacillus and Paenibacillus strains

  • These PGPR strains could provide complementary stress protection

Data from Field Studies:
Research has shown that B. japonicum strains vary in:

• Nitrogen fixation capacity

• Competitive ability for nodule occupancy

• Stress tolerance

The genomic variability found in natural B. japonicum variants adapted to different environments suggests that RecR functions may already be optimized for specific conditions, providing a natural resource for biotechnological improvements.

What new experimental techniques are emerging for studying RecR and homologous recombination in B. japonicum?

Several cutting-edge experimental techniques are emerging for studying RecR and homologous recombination in B. japonicum:

Advanced Imaging Technologies:

• Super-resolution microscopy:

  • Techniques like PALM, STORM, or STED for visualizing RecR localization

  • Single-molecule tracking of fluorescently labeled RecR in live cells

  • Observe real-time dynamics of RecFOR complex formation during DNA repair

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence imaging of RecR with ultrastructural context

  • Particularly valuable for studying RecR localization during bacteroid differentiation

  • Visualize RecR in relation to nucleoid organization in nodule cells

CRISPR-Based Technologies:

• CRISPR interference (CRISPRi):

  • Fine-tuned repression of recR expression

  • Study dosage effects without complete gene deletion

  • Allows temporal control of RecR depletion during symbiotic stages

• CRISPR-based genomic tagging:

  • Insert fluorescent or affinity tags at endogenous recR locus

  • Study native expression levels and localization patterns

  • Create libraries of tagged DNA repair proteins for interaction studies

Single-Cell Technologies:

• Single-cell RNA sequencing:

  • Analyze transcriptional heterogeneity in recR expression

  • Compare free-living cells vs. bacteroids within nodules

  • Correlate recR expression with other DNA repair and symbiotic genes

• Microfluidics-based approaches:

  • Track individual cell fates after DNA damage

  • Measure repair kinetics at single-cell resolution

  • Particularly valuable given B. japonicum's slow growth rate

Structural Approaches:

• Cryo-electron tomography:

  • Visualize RecFOR complexes in their native cellular context

  • Study the architecture of DNA repair centers in B. japonicum

  • Observe structural changes during symbiotic differentiation

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

  • Map protein dynamics and conformational changes

  • Identify RecR interaction surfaces with RecF, RecO, and DNA

  • Characterize allosteric regulation mechanisms

Multi-Omics Integration:

• Integrative analysis of transcriptomics, proteomics, and interactomics:

  • Construct comprehensive networks of RecR interactions

  • Similar to approaches used for B. diazoefficiens-soybean interactome

  • Identify novel components of DNA repair pathways in B. japonicum

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Map genome-wide binding sites of RecR and other recombination proteins

  • Identify hotspots for DNA repair activity

  • Correlate with genomic features like GC content and repetitive elements

These emerging techniques will provide unprecedented insights into RecR function in the context of B. japonicum's unique biology as a slow-growing, nitrogen-fixing symbiont with a large, GC-rich genome .

What are the major technical challenges in studying RecR function in B. japonicum?

Studying RecR function in B. japonicum presents several significant technical challenges:

Growth-Related Challenges:

• Extremely slow growth rate:

  • Generation time of B. japonicum (8-12 hours) significantly extends experimental timelines

  • Experiments that take days in E. coli require weeks in B. japonicum

  • Methodological solution: Develop optimized growth media that slightly accelerate growth while maintaining physiological relevance

• Contamination risks:

  • Extended incubation periods increase contamination probability

  • Solution: Use specialized selective media such as BJSM (B. japonicum selective medium)

  • Include appropriate antibiotics and antifungal agents in long-term cultures

Genetic Manipulation Difficulties:

• High spontaneous antibiotic resistance:

  • Complicates selection of true recombinants

  • Solution: Implement the rapid colony screening method described for B. japonicum

  • Use dual antibiotic selection strategies to reduce false positives

• Low transformation efficiency:

  • B. japonicum is notoriously difficult to transform

  • Solution: Optimize electroporation parameters specifically for B. japonicum

  • Consider alternative methods like conjugation with specialized E. coli donor strains

Protein Expression and Purification Issues:

• Codon usage bias:

  • High GC content (64.1%) of B. japonicum genome creates expression challenges

  • Solution: Use codon-optimized synthetic genes for heterologous expression

  • Consider specialized E. coli strains with rare tRNA supplementation

• Protein solubility and stability:

  • RecR may have evolved specific properties in B. japonicum

  • Solution: Screen multiple buffer conditions and consider fusion partners

  • Use solubility enhancers like SUMO tags and optimize purification protocols

Functional Assay Limitations:

• Distinguishing direct vs. indirect effects:

  • RecR functions as part of a complex network

  • Solution: Use in vitro reconstituted systems with purified components

  • Create partial function mutants rather than complete knockouts

• Symbiotic phenotype assessment:

  • Phenotypes may only manifest during symbiosis with soybean

  • Solution: Develop plant growth systems that allow controlled nodulation

  • Use fluorescent reporters to track bacteria within nodules

Comparison Table of Challenges and Solutions:

These technical challenges explain why RecR function in B. japonicum remains less characterized compared to model organisms, despite its agricultural importance.

How might our understanding of RecR in B. japonicum evolve with advances in systems biology?

Advances in systems biology are poised to revolutionize our understanding of RecR in B. japonicum through several integrative approaches:

Network-Based Understanding:

• Interactome mapping:

  • Application of techniques similar to those used for the B. diazoefficiens-G. max interactome

  • Identification of all proteins interacting with RecR beyond RecF and RecO

  • Integration of protein-protein and protein-DNA interaction networks

  • This would place RecR in its complete functional context

• Metabolic-repair pathway integration:

  • Connect DNA repair networks with nitrogen fixation metabolic pathways

  • Identify metabolic states that influence RecR function

  • Model how energy allocation to DNA repair affects symbiotic efficiency

  • This systems approach could explain why some B. japonicum strains show different symbiotic properties

Multi-Omics Integration:

• Layered data analysis:

  • Integrate transcriptomics, proteomics, metabolomics data

  • Apply machine learning to identify patterns in RecR regulation

  • Construct predictive models of RecR activity under different conditions

  • This approach could identify previously unknown regulatory factors

• Temporal dynamics modeling:

  • Track changes in RecR function throughout symbiotic stages

  • Model DNA repair system transitions during bacteroid differentiation

  • Create mathematical models of DNA damage and repair kinetics

  • This would address how RecR function changes during the symbiotic lifecycle

Evolutionary Systems Biology:

• Comparative genomic analysis:

  • Apply phylogenetic approaches similar to those used for multilocus sequence analysis

  • Reconstruct the evolutionary history of RecR across Bradyrhizobium species

  • Identify selective pressures on RecR in different ecological contexts

  • This could explain adaptation of repair systems to different host plants

• Pan-genome analysis:

  • Compare RecR sequence and function across the Bradyrhizobium pan-genome

  • Identify strain-specific adaptations in RecR and interacting partners

  • This approach could leverage the genetic variability found in B. japonicum and B. diazoefficiens strains

Synthetic Biology Applications:

• Minimal RecR module design:

  • Identify the core components required for RecR function

  • Engineer simplified RecFOR systems for controlled DNA repair

  • Create synthetic circuits linking RecR activity to symbiotic outputs

  • This could lead to biotechnological applications in agriculture

• Stress-responsive RecR systems:

  • Design synthetic genetic circuits that modulate RecR activity

  • Create feedback loops connecting DNA damage to repair system activation

  • This approach could enhance B. japonicum survival in agricultural settings

Predictive Modeling:
Based on systems biology data, computational models could predict:

• How RecR function affects symbiotic efficiency under different field conditions

• Optimal RecR expression levels for inoculant performance

• How genetic variations in RecR impact strain competitiveness and nitrogen fixation

These advances would transform our understanding of RecR from a simple DNA repair protein to a key component in an integrated system linking genomic integrity, symbiotic efficiency, and agricultural productivity.

What has genomic analysis revealed about RecR conservation across Bradyrhizobium species?

Recent genomic analyses have provided significant insights into RecR conservation patterns across Bradyrhizobium species:

Conservation Patterns:
Comparative genomic studies indicate that recR is part of the core genome in Bradyrhizobium species, showing higher conservation than many symbiotic genes. This conservation pattern reflects the essential nature of DNA repair functions across different ecological niches.

The multilocus sequence analysis (MLSA) approaches used to study Bradyrhizobium populations reveal that housekeeping genes (including DNA repair genes) generally show different evolutionary patterns compared to symbiotic genes:

Nucleotide Polymorphism Patterns:
Analysis of DNA polymorphisms in Bradyrhizobium populations using approaches similar to those described in search result would likely show:

• Lower nucleotide diversity (π) in recR compared to symbiotic genes

• Population genetic structures reflecting purifying selection on recR

• Limited recombination events affecting the recR locus compared to other regions

Genomic Context Conservation:
The complete genome sequence of B. japonicum USDA110 revealed that the 9.1 Mb chromosome contains a single set of essential repair genes . The recR gene is likely located in the conserved backbone of the chromosome rather than in the "symbiotic island" regions that show greater variability between strains.

Recent comparative genomic analysis of B. japonicum and B. diazoefficiens strains has identified significant genetic variation between closely related strains, including:

• Horizontal gene transfer events

• Genomic rearrangements

• Nucleotide polymorphisms

Taxonomic Implications:
The reclassification of some B. japonicum strains into B. diazoefficiens was based partly on molecular phylogeny of core genes. Given RecR's essential function, its sequence conservation versus divergence could provide valuable information for understanding the evolutionary relationships between Bradyrhizobium species and strains.

The genetic variability observed between B. japonicum and B. diazoefficiens groups, particularly in their pangenome size and nucleotide polymorphism frequency , suggests potential adaptation of DNA repair systems to different environmental conditions, which may include subtle adaptations in RecR function or regulation.

How do environmental stressors affect RecR function in B. japonicum?

Environmental stressors significantly impact RecR function in B. japonicum, with implications for both free-living survival and symbiotic performance:

Oxidative Stress Responses:
During nodule development and nitrogen fixation, B. japonicum encounters high levels of reactive oxygen species (ROS) that can damage DNA:

• RecR regulation under oxidative stress:

  • Expression of recR likely increases under oxidative conditions

  • The RecFOR pathway becomes critical for repairing oxidative DNA damage

  • This response is particularly important in bacteroids within nodules

• Methodological approach to study:

  • Measure recR expression in response to H₂O₂, paraquat, and other ROS generators

  • Assess survival of recR mutants under oxidative challenge

  • Quantify DNA damage (e.g., 8-oxoguanine levels) in wild-type vs. recR mutants

Temperature and Desiccation Effects:
B. japonicum inoculants face harsh temperature fluctuations and desiccation when applied to seeds:

• Impact on RecR function:

  • High temperature and desiccation decrease survival factors of B. japonicum on seeds

  • RecR-mediated DNA repair pathways are likely critical during these stresses

  • RecR protein stability may be affected by extreme conditions

• Experimental evidence:

  • Recovery experiments show exponential decay in bacterial viability under stress

  • Optimizing storage temperature improves survival

  • Osmoprotective compounds enhance recovery, possibly by supporting DNA repair functions

Soil Acidity and Metal Toxicity:
Many agricultural soils present acidic conditions and metal toxicity:

• RecR adaptation mechanisms:

  • B. japonicum strains isolated from acidic soils may have evolved specialized RecR variants

  • Metal ions (particularly Al³⁺) can induce DNA damage requiring RecR-dependent repair

  • pH fluctuations may affect RecR protein-protein interactions

• Practical implications:

  • Selection of B. japonicum strains with optimized RecR function for specific soil conditions

  • Development of inoculant formulations that protect DNA repair systems

Field-to-Laboratory Translation:
Studies comparing natural B. japonicum variants from different field conditions provide insights into environmental adaptation of DNA repair systems: