Recombinant Bradyrhizobium japonicum Ribonuclease PH (rph)

What is Bradyrhizobium japonicum and why is it significant in agricultural research?

Bradyrhizobium japonicum is a soil bacterium that forms symbiotic relationships with leguminous plants, particularly soybeans. Its agricultural significance stems from its ability to fix atmospheric nitrogen through specialized root nodule structures. The bacteria contain Bradyrhizobium that can take nitrogen from the air and convert it into forms that plants can use, effectively serving as a natural fertilizer . For optimal nitrogen fixation in fields where soybeans haven't been cultivated recently, farmers should inoculate seeds with Bradyrhizobium japonicum, typically applied as a seed coating . This symbiotic relationship reduces the need for chemical nitrogen fertilizers, contributing to sustainable agricultural practices.

What is Ribonuclease PH and what role might it play in Bradyrhizobium japonicum?

Ribonuclease PH (rph) is a phosphorolytic exoribonuclease involved in RNA processing, particularly in the maturation of transfer RNA (tRNA) and ribosomal RNA (rRNA). While specific information about its function in B. japonicum is limited in the provided search results, the enzyme likely plays critical roles in RNA turnover and quality control, similar to its function in other bacteria. In B. japonicum, which undergoes significant physiological changes during the transition from free-living to symbiotic states, rph may be particularly important for regulating gene expression during these transitions through its RNA processing activities.

How does the nitrogen fixation process in Bradyrhizobium japonicum operate at the molecular level?

Nitrogen fixation in B. japonicum involves complex regulatory networks, including the NtrBC two-component system, which is "a critical regulator of cellular nitrogen metabolism, including the acquisition and catabolism" of nitrogen compounds . The bacteria form root nodules where they convert atmospheric N₂ to ammonia using the nitrogenase enzyme complex, which requires significant energy input and specialized oxygen-limited conditions. This process is tightly regulated, with numerous genes involved in nodule formation, nitrogen fixation, and metabolic adjustments. Co-inoculation studies show that nodule nitrogenase activity can be significantly enhanced when B. japonicum is paired with other beneficial bacteria like Bacillus aryabhattai .

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

Based on the search results, successful expression of recombinant proteins from Bradyrhizobium can be achieved using the pET-21d(+) expression system in E. coli . When working with B. japonicum proteins, researchers should consider the following methodological aspects:

• Codon optimization may be necessary due to B. japonicum's high GC content genome, which differs from E. coli's codon usage patterns.

• Expression strain selection is critical - non-suppressor strains like HB2151 have been successfully used for soluble expression of Bradyrhizobium-related proteins .

• Induction conditions require careful optimization, including temperature, inducer concentration, and duration.

• Purification strategies should incorporate appropriate affinity tags while maintaining protein functionality.

• Post-purification verification of enzymatic activity is essential to confirm the recombinant protein retains its native function.

What genetic manipulation techniques are most effective for studying gene function in B. japonicum?

Search result describes a comprehensive methodology for creating deletion mutants in B. japonicum:

• PCR amplification of genomic DNA including approximately 500 bp of 5' and 3' flanking sequences of the target gene using high-fidelity DNA polymerase.

• Cloning the amplified fragments into appropriate vectors (e.g., pKOTc or pKOTc2).

• Generation of a kanamycin resistance cassette flanked by FRT (flippase recognition target) sites.

• Homologous recombination using the λ Red system in E. coli.

• Transfer of the deletion construct to B. japonicum via triparental mating with helper plasmid pRK2073.

• Selection of double-recombination mutants based on kanamycin resistance and tetracycline sensitivity.

• Optional removal of the kanamycin marker using FLP recombinase, allowing for subsequent genetic modifications .

This approach enables precise gene deletion for functional characterization of targets like rph.

What methods are available for detecting and monitoring B. japonicum in experimental systems?

Several sophisticated detection methods for Bradyrhizobium are described in the search results:

• Recombinant antibody-based detection: Single-chain variable fragments (scFv) generated through phage display technology can specifically detect Bradyrhizobium strains in both symbiotic and endophytic contexts .

• ELISA (Enzyme-Linked Immunosorbent Assay): Provides quantitative measurement of Bradyrhizobium populations using specific antibodies .

• Confocal immunofluorescence imaging: Enables visualization of bacteria within plant tissues with high specificity and spatial resolution .

• Reporter gene systems: The β-glucuronidase (GUS) reporter system serves as a standard method for tracking tagged Bradyrhizobium strains in nodule occupancy studies .

These methodologies could be adapted to study the expression and localization of rph in different physiological states of B. japonicum.

How might RNA processing via Ribonuclease PH contribute to symbiotic adaptation in B. japonicum?

Although the search results don't directly address the role of Ribonuclease PH in B. japonicum symbiosis, RNA processing likely plays a critical role in the bacterium's transition from free-living to symbiotic states. Methodological approaches to investigate this question could include:

• Comparative transcriptomics of wild-type and rph mutant strains during different stages of symbiosis to identify differentially processed RNAs.

• In vitro RNA processing assays using recombinant rph to determine substrate specificity under different physiological conditions.

• CLIP-seq (Crosslinking Immunoprecipitation Sequencing) to identify the direct RNA targets of rph during symbiotic development.

• Phenotypic analysis of rph mutants focusing on nodulation efficiency, nitrogen fixation rates, and competitiveness against wild-type strains.

This research could reveal how post-transcriptional regulation via RNA processing contributes to the complex developmental changes required for successful symbiosis.

What influence does bacterial co-inoculation have on gene expression in B. japonicum?

Search result provides significant insights into this question, demonstrating that co-inoculation of B. japonicum with other beneficial bacteria substantially affects both soil properties and plant growth. Metagenomic sequencing revealed that:

• Co-inoculation with Bacillus aryabhattai (designated as RB treatment) significantly improved:

  • Nodule nitrogenase activity

  • Soil nitrogen content and urease activity

  • Abundance of nitrogen cycle genes

  • Specific bacterial populations in the rhizosphere (Betaproteobacteria and Chitinophagia)

• Co-inoculation with Paenibacillus mucilaginosus (RP treatment) significantly affected:

  • Phosphorus cycling gene abundance

  • Soil available phosphorus and phosphatase activity

  • Different bacterial populations (Deltaproteobacteria and Gemmatimonadetes)

• Triple inoculation with all three strains (RBP) produced the greatest benefits for plant growth:

  • Increased soybean nitrogen content

  • Enhanced plant dry weight

  • Coordinated modulation of the rhizosphere microbial community

These findings suggest complex interactions between the introduced bacterial strains that may involve signaling networks, metabolic complementation, and altered gene expression patterns.

How does the NtrBC regulatory system interact with RNA processing in B. japonicum?

The NtrBC two-component system is identified as a critical regulator of nitrogen metabolism in B. japonicum . While direct interactions with RNA processing machinery aren't explicitly described in the search results, potential methodological approaches to investigate this connection include:

• Comparative transcriptomic analysis of wild-type versus ntrBC mutants, focusing on changes in RNA processing patterns.

• Examination of rph expression levels in response to NtrBC activity under different nitrogen conditions.

• Chromatin immunoprecipitation sequencing (ChIP-seq) to determine if NtrC directly regulates genes encoding RNA processing enzymes.

• Protein-protein interaction studies to identify potential physical associations between NtrBC components and RNA processing machinery.

Table 1 below summarizes genes regulated by NtrC based on data from search result , which could provide insights into potential regulatory connections with RNA processing:

What are common challenges in expressing and purifying enzymatically active recombinant B. japonicum proteins?

While the search results don't specifically address recombinant Ribonuclease PH from B. japonicum, several general challenges and methodological solutions can be anticipated:

• Codon usage optimization: B. japonicum's high GC content genome presents challenges for heterologous expression. Methodological approaches include synthetic gene design with codon optimization or using specialized E. coli strains carrying rare tRNAs.

• Protein solubility issues: Recombinant expression often results in inclusion body formation. Effective methodological strategies include:

  • Reduced-temperature expression protocols (16-20°C)

  • Fusion with solubility-enhancing tags (MBP, SUMO, etc.)

  • Co-expression with molecular chaperones

  • Expression in specialized E. coli strains designed for difficult proteins

• Maintaining enzymatic activity: RNA processing enzymes require specific conditions to retain functionality. Methodological considerations include:

  • Buffer optimization during purification steps

  • Addition of stabilizing agents (glycerol, reducing agents)

  • Limited exposure to freeze-thaw cycles

  • Activity assays using physiologically relevant substrates

• Protein yield optimization: Low expression levels can be addressed through:

  • Promoter selection and induction protocol optimization

  • Media composition adjustments

  • Scale-up strategies with controlled growth parameters

What factors influence successful genetic manipulation of B. japonicum?

Based on the detailed protocols in search result , several methodological factors affect genetic manipulation efficiency in B. japonicum:

• Homology length: The methodology described uses approximately 500 bp of flanking sequences for homologous recombination, indicating this is an effective length for successful recombination events in B. japonicum .

• Selection strategy: The methodology employs a dual-selection approach (kanamycin resistance for positive selection and tetracycline sensitivity for counter-selection), which increases specificity .

• Transfer methodology: Triparental mating with a helper plasmid (pRK2073) is the preferred method for introducing constructs into B. japonicum .

• Confirmation protocols: Multiple verification steps are necessary, including PCR confirmation with primers spanning the expected deletion junctions and antibiotic sensitivity testing .

• Marker removal system: The FRT/FLP system allows for marker removal, enabling the construction of multiple mutations in a single strain .

These methodological considerations are directly applicable to creating rph deletion mutants for functional studies.

How can researchers optimize enzymatic activity assays for Ribonuclease PH?

While specific assays for B. japonicum Ribonuclease PH aren't described in the search results, methodological approaches for ribonuclease activity assessment could include:

• Substrate selection: Synthetic RNA oligonucleotides with defined structures that mimic physiological substrates (tRNA precursors, rRNA fragments) provide controlled conditions for activity measurement.

• Detection methods:

  • Radioisotope-labeled substrates followed by gel electrophoresis and phosphorimaging

  • Fluorescence-based assays using fluorophore-quencher labeled RNAs

  • HPLC or mass spectrometry analysis of reaction products

  • Colorimetric assays measuring phosphate release

• Reaction condition optimization:

  • Buffer composition (pH, ionic strength, divalent cations)

  • Temperature ranges relevant to B. japonicum's lifecycle

  • Inorganic phosphate concentration (as rph requires phosphate for activity)

  • Potential inhibitors or enhancers

• Control reactions:

  • Heat-inactivated enzyme controls

  • RNase-free conditions to prevent contamination

  • Known ribonuclease inhibitors as specificity controls

How might understanding Ribonuclease PH function contribute to enhancing B. japonicum's agricultural applications?

RNA processing enzymes like Ribonuclease PH represent potential targets for enhancing B. japonicum's beneficial properties. Future research directions could include:

• Engineering strains with modified rph expression to optimize RNA processing for specific agricultural conditions.

• Investigating how rph activity correlates with symbiotic efficiency and nitrogen fixation rates.

• Developing rph-based molecular markers for tracking high-performing B. japonicum strains in field applications.

• Exploring how environmental stressors affect rph function and whether enhanced RNA processing could improve stress tolerance.

The co-inoculation studies in search result demonstrate that bacterial combination approaches significantly enhance beneficial effects, suggesting that engineered B. japonicum with optimized RNA processing could further improve these benefits.

What novel methodologies are emerging for studying RNA processing in symbiotic bacteria?

Although not directly addressed in the search results, cutting-edge methodologies that could be applied to studying RNA processing in B. japonicum include:

• Nanopore direct RNA sequencing: Enables detection of RNA modifications and processing events without conversion to cDNA.

• Ribosome profiling: Provides insights into how RNA processing affects translation efficiency.

• CRISPR-Cas9 based approaches: Allows precise genetic manipulation of RNA processing components.

• Single-cell RNA-seq: Reveals heterogeneity in bacterial populations during symbiotic interactions.

• Structural biology techniques (cryo-EM): Provide atomic-level insights into enzyme-substrate interactions.

How might the study of RNA processing enzymes like Ribonuclease PH intersect with biofertilizer development?

Search result highlights that Bradyrhizobium is "under investigation as an efficient biofertilizer for sustainable legume-rice rotational cropping system." Understanding RNA processing in this context could contribute to biofertilizer development through:

• Identification of RNA processing signatures associated with high-performing biofertilizer strains.

• Engineering of RNA processing machinery to enhance bacterial survival in commercial formulations.

• Development of RNA-based monitoring tools for tracking biofertilizer performance in field applications.

• Optimization of gene expression patterns through targeted RNA processing to improve nitrogen fixation efficiency.

The methodology described for generating specific antibodies against Bradyrhizobium strains could be adapted to develop detection tools specific for engineered strains with modified RNA processing capabilities .