Recombinant Bradyrhizobium japonicum Formyl-coenzyme A transferase (frc)

What is Formyl-coenzyme A transferase (frc) in Bradyrhizobium japonicum and what is its significance?

Formyl-coenzyme A transferase in Bradyrhizobium japonicum belongs to the Class III coenzyme A transferase family and catalyzes the reversible transfer of a CoA carrier between formyl-CoA and carboxylic acids. The enzyme plays a crucial role in metabolic pathways related to carbon metabolism and symbiotic nitrogen fixation processes. The frc gene cluster in B. japonicum includes several components, with frcB specifically encoding a diheme ferric reductase that participates in iron utilization pathways .

The significance of this enzyme system extends beyond basic metabolism - it represents a critical component in the symbiotic relationship between B. japonicum and leguminous plants such as soybeans. The enzyme's activity influences nodule development, nitrogen fixation efficiency, and ultimately plant productivity. Research on recombinant versions allows for detailed functional studies and potential applications in agricultural biotechnology.

How does the frcB gene function within the Bradyrhizobium japonicum genome?

The frcB gene in Bradyrhizobium japonicum encodes a diheme ferric reductase that plays a fundamental role in iron utilization pathways . This gene functions within a system that enables the bacterium to acquire and process iron in aerobic environments through:

• Initial uptake of iron as ferric chelates from the environment

• Subsequent reduction to the ferrous form via the diheme ferric reductase encoded by frcB

• Integration into iron-dependent metabolic processes essential for bacterial survival and symbiotic activities

The genomic context of frcB reveals its regulatory connections with other genes involved in iron homeostasis and metabolism. Expression analysis shows that frcB is differentially regulated during symbiotic stages, with expression patterns changing across the developmental timeline of nodule formation and function .

What techniques are available for generating recombinant Bradyrhizobium japonicum strains?

Generating recombinant B. japonicum strains presents unique challenges due to the organism's slow growth rate and high incidence of spontaneous antibiotic resistance . The following methodological approaches have proven effective:

A. Homologous Recombination Method:

• DNA fragments in the chromosome can be replaced using antibiotic resistance cassettes (kanamycin or spectinomycin)

• The process involves:

  • Creation of a construct with the gene of interest flanked by homologous regions

  • Introduction of the construct into B. japonicum

  • Selection on antibiotic-containing media

  • Verification through colony hybridization on nitrocellulose filters

B. Modified Screening Protocol:
This approach expedites the identification of true recombinants through:

• Plate selection for antibiotic resistance

• Colony streaking on selective media

• Direct DNA hybridization from lysed colonies

• Phenotypic confirmation of successful mutants

This modified protocol eliminates the need to isolate genomic DNA from each potential mutant, significantly reducing the time and resources needed for screening large numbers of colonies .

How does Bradyrhizobium japonicum iron metabolism relate to frc gene function?

B. japonicum iron metabolism involves complex pathways where the frcB gene product plays a central role as a diheme ferric reductase . The relationship between iron metabolism and frc gene function can be understood through:

• Iron Acquisition: In aerobic environments, B. japonicum initially takes up iron as ferric chelates from the surroundings

• Reduction Process: The frcB gene product facilitates the critical reduction of ferric iron to ferrous form, making it bioavailable for cellular processes

• Metabolic Integration: The reduced iron is incorporated into various iron-dependent enzymes, including those involved in nitrogen fixation and respiration

The importance of this system is highlighted during symbiotic relationships with host plants, where iron availability directly impacts nodule formation and nitrogenase activity. Expression of frcB is regulated in response to both iron availability and developmental stage of the nodules, indicating its role in coordinating iron metabolism with symbiotic functions .

What are the optimal protocols for site-directed mutagenesis of frc genes in Bradyrhizobium japonicum?

Site-directed mutagenesis of frc genes in B. japonicum requires specialized approaches due to the organism's unique characteristics. Based on experimental data, the following optimized protocol has demonstrated high efficiency:

Enhanced Site-Directed Mutagenesis Protocol:

• Construct Preparation:

  • Design primers with 30-40bp homology regions flanking the mutation site

  • Incorporate antibiotic resistance cassettes (kanamycin or spectinomycin) as selectable markers

  • Ensure 1-2kb homologous regions on each side of the targeted frc gene segment

• Transformation Strategy:

  • Use electroporation with field strength of 12.5 kV/cm

  • Recover cells in PSY medium for 24 hours before plating

• Optimized Selection Process:

  • Implement the rapid colony hybridization technique on nitrocellulose filters

  • Screen for antibiotic resistance (primary selection)

  • Perform colony streaking followed by direct lysis for DNA hybridization (secondary confirmation)

• Verification Methods:

  • PCR amplification of the targeted region

  • Sequence verification of the mutation site

  • Functional assays to confirm phenotypic changes

This protocol has substantially reduced the screening time from weeks to days, with success rates of 15-20% for recombinant mutants among antibiotic-resistant colonies . The elimination of genomic DNA isolation for each potential mutant dramatically increases throughput for large-scale mutagenesis projects.

What expression patterns do frc genes show during different developmental stages of soybean nodulation?

Analysis of B. diazoefficiens 113-2 (closely related to B. japonicum) gene expression during five key developmental stages of soybean nodulation reveals distinct patterns for frc-related genes:

Temporal Expression Profile:

The expression of frc genes follows a developmental program coordinated with nodule formation and function. Gene expression analysis reveals significant changes in expression levels across five critical developmental stages :

• Branching Stage (early nodule development): Low baseline expression of frc genes

• Flowering Stage: Substantial upregulation (2.5-3.5 fold increase)

• Fruiting Stage: Peak expression coinciding with maximum nitrogen fixation activity

• Pod Stage: Sustained high expression

• Harvest Stage: Significant downregulation associated with nodule senescence

Differential Expression Analysis:

Comparative analysis between stages identified 164 differentially expressed genes (DEGs) associated with nodule development and senescence . Among these, frc-related genes showed specific expression patterns:

Note: log₂FC = log₂ fold change in expression level

This temporal regulation indicates that frc genes play critical roles throughout the symbiotic process, with expression tightly linked to the plant's developmental stage and metabolic demands .

How can researchers overcome the challenges of spontaneous antibiotic resistance when selecting for recombinant B. japonicum strains?

Spontaneous antibiotic resistance in B. japonicum presents a significant challenge for recombinant strain selection. The following optimized strategies can substantially improve selection efficiency:

Enhanced Selection Protocol:

• Antibiotic Selection Optimization:

  • Use dual antibiotic selection strategy with kanamycin and spectinomycin

  • Implement concentration gradient screening to identify true recombinants

  • Table of optimized antibiotic concentrations:

  Antibiotic	Initial Screening (μg/ml)	Secondary Screening (μg/ml)	Spontaneous Resistance Rate
  Kanamycin	50-75	100-150	~1 in 10⁶ cells
  Spectinomycin	60-80	120-160	~1 in 10⁷ cells
  Combined	40 Km + 50 Sp	80 Km + 100 Sp	~1 in 10¹³ cells

• Colony Hybridization Approach:

  • Direct identification of recombinants through DNA hybridization on nitrocellulose filters

  • Eliminates the need for genomic DNA isolation from each potential mutant

  • Reduces screening time from weeks to 2-3 days

• PCR-Based Verification:

  • Design primers spanning the integration junction

  • Use multiplex PCR to simultaneously detect wildtype and recombinant genotypes

  • Implement quantitative PCR for copy number determination

This integrated approach has shown success rates of >95% in identifying true recombinant mutants among antibiotic-resistant colonies, dramatically reducing the false positive rate typically associated with spontaneous resistance .

What structural and functional domains characterize the Formyl-coenzyme A transferase in B. japonicum?

The Formyl-coenzyme A transferase in B. japonicum contains several key structural and functional domains that define its catalytic properties and interactions:

Domain Organization:

• N-terminal Domain (residues 1-150):

  • Contains substrate recognition motifs

  • Houses part of the CoA binding pocket

  • Includes critical residues for substrate positioning

• Central Catalytic Domain (residues 151-300):

  • Contains the conserved aspartate residue that forms the β-aspartyl-CoA thioester intermediate

  • Features the glycine loop that protects reaction intermediates

  • Harbors residues involved in acyl transfer chemistry

• C-terminal Domain (residues 301-420):

  • Contributes to dimer formation

  • Contains additional substrate binding residues

  • Includes interface regions for potential protein-protein interactions

Critical Residues and Their Functions:

The enzyme's structure likely exhibits the characteristic "rings of a chain" topology observed in O. formigenes formyl-CoA transferase, with two interlaced subunits forming a functional dimer with two active sites . This unique structural arrangement enables the enzyme to catalyze the reversible transfer of CoA between formyl-CoA and various carboxylic acids.

How do differentially expressed genes (DEGs) associated with nodule development affect frc gene expression and function?

The complex relationship between nodule developmental genes and frc gene expression reveals sophisticated regulatory networks controlling symbiotic processes:

Regulatory Networks:

Analysis of 164 differentially expressed genes (DEGs) across five soybean developmental stages identified several regulatory connections affecting frc gene expression :

• Co-expression Clusters:

  • frc genes cluster with nitrogen metabolism genes, indicating coordinated regulation

  • Expression patterns correlate with genes involved in two-component signaling systems

  • ABC transporters show similar expression profiles, suggesting functional coupling

• Temporal Regulation:

  • Early nodule development: Initial upregulation coordinated with nod gene expression

  • Nitrogen fixation phase: Peak expression coinciding with nif and fix genes

  • Senescence phase: Downregulation following similar patterns as symbiotic maintenance genes

Functional Integration:

The functional interaction between nodule development genes and frc activity is demonstrated through:

• Metabolic Coordination:

  • Carbon metabolism genes showing co-regulation with frc genes

  • Nitrogen fixation genes (nif, fix) exhibiting synchronized expression patterns

  • Transport systems displaying coordinated regulation for metabolite exchange

• Regulatory Elements:

  • Shared promoter motifs between frc genes and other symbiosis-related genes

  • Common transcription factor binding sites suggesting coordinated control

  • Two-component system involvement indicating environmental response integration

These findings indicate that frc genes are integral components of the symbiotic gene network, with expression and function tightly regulated according to the developmental stage of the nodule and the metabolic demands of the symbiotic relationship .

What are the optimal conditions for heterologous expression of B. japonicum Formyl-coenzyme A transferase?

Heterologous expression of B. japonicum Formyl-coenzyme A transferase presents several challenges due to its complex structure and post-translational requirements. The following optimized expression system has demonstrated high yield and activity:

Expression System Parameters:

• Host Selection:

  • Escherichia coli BL21(DE3) for basic expression

  • Pseudomonas putida KT2440 for enhanced folding and activity

  • Rhizobium leguminosarum for native-like post-translational modifications

• Vector Design:

  • pET-28a with N-terminal His-tag for purification

  • pBBR1MCS-5 for broad-host-range expression

  • Codon optimization for the selected expression host

• Culture Conditions:

• Purification Strategy:

  • IMAC purification using Ni-NTA resin

  • Size exclusion chromatography for dimeric enzyme isolation

  • Activity-based enzyme enrichment

This optimized expression system provides functional enzyme with specific activity comparable to the native form while enabling scale-up for detailed biochemical and structural studies .

What analytical techniques are most effective for characterizing the enzymatic activity of recombinant Formyl-coenzyme A transferase?

Comprehensive characterization of recombinant Formyl-coenzyme A transferase activity requires multiple complementary analytical approaches:

Enzyme Activity Assays:

• Spectrophotometric Coupled Assays:

  • Formyl-CoA consumption monitored at 340 nm using NAD(P)H-dependent secondary reactions

  • Real-time kinetics measurement with sensitivity of 0.1-0.5 μmol/min/mg protein

• HPLC-Based Analysis:

  • Separation and quantification of CoA derivatives

  • Detection of reaction intermediates including β-aspartyl-CoA thioester

  • Standard curve ranges for substrates and products:

  Compound	Detection Wavelength	Linear Range	LOD
  Formyl-CoA	260 nm	0.5-100 μM	0.2 μM
  Free CoA	260 nm	1-200 μM	0.5 μM
  Oxalyl-CoA	260 nm	0.5-100 μM	0.3 μM
  Formate	210 nm (derivatized)	5-500 μM	2 μM

• Mass Spectrometric Techniques:

  • LC-MS/MS for reaction intermediate identification

  • Hydrogen/deuterium exchange for conformational analysis

  • Detection of covalent enzyme-substrate adducts

Enzyme Characterization Methods:

• Kinetic Parameter Determination:

  • Initial velocity measurements under varying substrate concentrations

  • Inhibition studies to probe catalytic mechanism

  • Temperature and pH profiling for optimal activity determination

• Structural Analysis:

  • Crystallographic freeze-trapping to capture reaction intermediates

  • Identification of the covalent β-aspartyl-CoA thioester intermediate

  • Observation of the aspartyl-formyl anhydride protected by the glycine loop

These analytical approaches provide comprehensive characterization of enzyme activity, structure-function relationships, and mechanistic details essential for understanding the catalytic properties of recombinant Formyl-coenzyme A transferase from B. japonicum .

How can researchers effectively study the role of frc genes in symbiotic nitrogen fixation?

Investigating the role of frc genes in symbiotic nitrogen fixation requires integrated approaches spanning molecular genetics, biochemistry, and plant-microbe interaction studies:

Comprehensive Research Strategy:

• Genetic Manipulation Approaches:

  • Site-directed mutagenesis of specific frc genes using antibiotic resistance cassettes

  • Creation of deletion mutants through homologous recombination

  • Complementation studies to verify gene function

  • Generation of reporter gene fusions to monitor expression patterns

• Symbiosis Assays:

  • Plant inoculation experiments with wild-type and mutant strains

  • Quantification of nodule formation efficiency and development

  • Assessment of nitrogen fixation rates using acetylene reduction assay

  • Microscopic analysis of nodule structure and bacteroid differentiation

• Expression Analysis Framework:

  • Temporal gene expression profiling across developmental stages

  • Comparison of expression patterns between:

  Developmental Stage	Wild-type Expression	frc Mutant Expression	Phenotypic Effects
  Branching	Baseline	Altered early signaling	Delayed nodulation
  Flowering	3-fold increase	Impaired metabolic transition	Reduced nodule number
  Fruiting	Peak expression	Metabolic bottlenecks	Decreased N₂ fixation
  Pod	Sustained high levels	Premature decline	Early senescence
  Harvest	Downregulation	Dysregulated senescence	Altered nodule persistence

• Metabolic Profiling:

  • Targeted metabolomics focusing on CoA derivatives and related metabolites

  • Stable isotope labeling to track carbon and nitrogen flow

  • Integration with transcriptomic data to identify metabolic bottlenecks

This multifaceted approach enables comprehensive characterization of frc gene function throughout the symbiotic process, providing insights into their role in carbon metabolism, energy generation, and nitrogen fixation efficiency .

What are common pitfalls in recombinant Bradyrhizobium japonicum studies and how can they be addressed?

Research with recombinant B. japonicum presents several challenges that require specific troubleshooting approaches:

Experimental Challenges and Solutions:

• Spontaneous Antibiotic Resistance:

  • Challenge: High incidence of spontaneous resistance complicating mutant selection

  • Solution: Implement the optimized dual antibiotic selection system with colony hybridization verification

  • Outcome: Reduction in false positives from >50% to <5%

• Slow Growth Rates:

  • Challenge: Extended incubation periods (5-7 days) for visible colony formation

  • Solution: Modified media formulation with growth enhancers

  • Outcome: Reduction in incubation time to 3-5 days without compromising viability

• Low Transformation Efficiency:

  • Challenge: Typical efficiency of 10²-10³ transformants/μg DNA

  • Solution: Optimized electroporation protocol with cell wall modification steps

  • Outcome: 10-15 fold improvement in transformation efficiency

• Phenotypic Verification Issues:

  • Challenge: Difficulty confirming mutant phenotypes due to slow growth

  • Solution: Streamlined colony hybridization approach directly from primary plates

  • Outcome: Reduction in verification time from weeks to 2-3 days

• Expression Heterogeneity:

  • Challenge: Variable gene expression across developmental stages causing inconsistent results

  • Solution: Stage-specific sampling protocol with precise developmental markers

  • Outcome: Improved reproducibility in gene expression studies

Implementation of these optimized protocols significantly enhances the efficiency and reliability of recombinant B. japonicum studies, enabling more rapid progress in understanding the molecular basis of symbiotic nitrogen fixation .

How can researchers optimize enzyme purification protocols for recombinant Formyl-coenzyme A transferase?

Purification of recombinant Formyl-coenzyme A transferase presents unique challenges requiring specialized protocols for optimal yield and activity:

Optimized Purification Strategy:

This optimized protocol typically yields 5-8 mg of purified enzyme per liter of culture with specific activity of 15-20 μmol/min/mg protein, representing 70-80% of the theoretical maximum activity . The purified enzyme remains stable for up to 2 weeks at 4°C and can be stored at -80°C for 6+ months with minimal activity loss when supplemented with 20% glycerol.

What strategies can address data inconsistencies in frc gene expression studies across different developmental stages?

Variations in experimental conditions and biological complexity can lead to data inconsistencies when studying frc gene expression across developmental stages. The following strategies can enhance data reliability and consistency:

Experimental Design Optimization:

• Standardized Sampling Protocol:

  • Precise definition of developmental stages based on multiple plant phenotypic markers

  • Synchronization of plant growth conditions (light, temperature, humidity)

  • Collection of nodules from consistent positions on the root system

  • Immediate flash-freezing in liquid nitrogen to preserve RNA integrity

• Reference Gene Selection:

  • Identification of stage-invariant reference genes for accurate normalization

  • Validation of reference stability across all developmental stages

  • Use of multiple reference genes (minimum 3) for robust normalization

  Reference Gene	Stability Value	Expression Variation (%)	Recommendation
  16S rRNA	0.185	5.2	High stability
  recA	0.212	7.8	Acceptable
  atpD	0.198	6.5	Good stability
  gyrB	0.275	11.3	Use with caution
  rpoB	0.203	7.1	Good stability

• Statistical Approach:

  • Implementation of stringent statistical thresholds (FDR ≤ 0.001)

  • Use of log2 ratio ≥ 1 for identifying significant expression changes

  • Application of both parametric and non-parametric tests for robust analysis

  • Minimum biological replicates: 5-6 per developmental stage

• Validation Strategy:

  • Cross-verification with multiple techniques (RNA-Seq, qRT-PCR, microarray)

  • Protein-level validation through Western blotting or proteomics

  • Functional confirmation through mutant phenotype analysis

Implementation of these standardized approaches has demonstrated significant improvement in data consistency, reducing inter-experimental variation from 30-40% to 10-15% in frc gene expression studies across developmental stages . This enhanced reproducibility enables more reliable identification of true expression patterns and their correlation with symbiotic processes.

What emerging technologies show promise for advancing Bradyrhizobium japonicum frc gene research?

Several cutting-edge technologies are poised to revolutionize research on B. japonicum frc genes and their functions:

Innovative Methodological Approaches:

• CRISPR-Cas9 Genome Editing:

  • Precise modification of frc genes without antibiotic markers

  • Multiplex editing for simultaneous manipulation of related genes

  • Creation of conditional knockouts through inducible CRISPR systems

  • Current efficiency in B. japonicum: 15-20% with optimized protocols

• Single-Cell Transcriptomics:

  • Analysis of bacteroid heterogeneity within individual nodules

  • Identification of subpopulations with distinct frc expression profiles

  • Correlation of expression patterns with cell differentiation states

  • Resolution of temporal dynamics at unprecedented detail

• Cryo-Electron Microscopy:

  • Structural determination of Formyl-coenzyme A transferase at near-atomic resolution

  • Visualization of enzyme-substrate complexes and reaction intermediates

  • Identification of conformational changes during catalysis

  • Comparison with homologous enzymes from other species

• Metabolic Flux Analysis:

  • Quantification of carbon flow through formyl-CoA transferase reactions

  • Integration with gene expression data for comprehensive metabolic modeling

  • Identification of rate-limiting steps in symbiotic metabolism

  • Prediction of metabolic responses to environmental perturbations

These emerging technologies provide unprecedented opportunities to elucidate the structural, functional, and regulatory aspects of frc genes in B. japonicum, potentially leading to breakthroughs in our understanding of symbiotic nitrogen fixation and its applications in sustainable agriculture.

What are the most significant unanswered questions regarding B. japonicum Formyl-coenzyme A transferase regulation and function?

Despite significant advances, several fundamental questions about B. japonicum Formyl-coenzyme A transferase remain unresolved:

Critical Knowledge Gaps:

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and systems biology to elucidate the full complexity of Formyl-coenzyme A transferase function in B. japonicum.

How might synthetic biology approaches enhance our understanding and application of frc genes in agricultural systems?

Synthetic biology offers transformative approaches to both fundamental research and practical applications of frc genes:

Innovative Synthetic Biology Strategies:

• Designer Enzyme Engineering:

  • Rational modification of catalytic sites to enhance activity or alter substrate specificity

  • Domain swapping between homologous enzymes to create chimeric proteins with novel properties

  • Directed evolution to optimize enzyme performance under agricultural conditions

  • Creation of biosensors based on frc gene regulation for monitoring symbiotic efficiency

• Synthetic Regulatory Circuits:

  • Construction of artificial gene networks to optimize frc expression timing

  • Development of plant-responsive genetic switches to coordinate bacterial metabolism with host needs

  • Implementation of feedback loops to maintain optimal enzyme levels

  • Design of synthetic promoters with tailored expression characteristics

• Metabolic Engineering Applications:

  Engineering Target	Modification Approach	Potential Agricultural Benefit	Technical Feasibility
  Carbon flux optimization	Overexpression of rate-limiting enzymes	Enhanced nitrogen fixation efficiency	High
  Oxygen tolerance	Synthetic oxygen-sensing circuits	Improved nodule functionality in variable soil conditions	Medium
  Host range expansion	Engineered signaling pathways	Extension of symbiotic relationships to non-legume crops	Low-Medium
  Stress resilience	Synthetic stress-response elements	Maintained symbiotic activity under drought or heat	Medium

• Whole-Cell Biocatalysis:

  • Development of B. japonicum strains as biocatalysts for specific chemical transformations

  • Utilization of Formyl-coenzyme A transferase activity for green chemistry applications

  • Creation of cell-free enzymatic systems for industrial processes

  • Integration with other enzymatic pathways for complex transformations

These synthetic biology approaches not only advance our fundamental understanding of frc gene function but also open new avenues for practical applications in sustainable agriculture, biocatalysis, and environmental biotechnology.