Recombinant Bradyrhizobium japonicum Transcription elongation factor GreA (greA)

What is Bradyrhizobium japonicum and why is it important in research?

Bradyrhizobium japonicum is a soil bacterium belonging to the α-Proteobacteria group. It has significant agricultural importance as it forms nitrogen-fixing symbiotic relationships with legumes, particularly soybeans. Research has demonstrated that B. japonicum strains, such as USDA6 and E109, exhibit distinct physiological characteristics including relatively fast growth rates compared to related species like B. diazoefficiens . Beyond nitrogen fixation, certain strains (e.g., FCBP-SB-406) have shown remarkable potential as biocontrol agents against soil-borne pathogens like Macrophomina phaseolina, reducing disease severity by up to 81.25% in controlled studies . This dual role in plant growth promotion and disease suppression makes B. japonicum a valuable subject for agricultural microbiology research.

What is the function of transcription elongation factor GreA in bacteria?

Transcription elongation factor GreA belongs to a family of prokaryotic proteins that play crucial roles in transcriptional regulation. This factor facilitates RNA polymerase progression during transcription by helping resolve paused or arrested elongation complexes. In molecular terms, GreA promotes the hydrolytic cleavage of nascent RNA in backtracked transcription complexes, allowing transcription to resume efficiently. The protein contains two consensus prokaryotic transcription elongation factor signatures (Prosite PS00829 and PS00830) that are conserved across bacterial species . In rhizobia, GreA appears in genomic arrangements with LPS biosynthesis genes, suggesting potential co-regulation or functional relationships between transcription processes and cell envelope biosynthesis, which may be particularly relevant during host-microbe interactions.

How is the greA gene organized in the Bradyrhizobium japonicum genome?

While the specific organization in B. japonicum isn't directly detailed in the provided sources, comparative genomic analysis of related rhizobia provides insights. In Sinorhizobium meliloti, the greA gene is contiguous with and transcribed in the same direction as lpsB, which encodes an enzyme involved in lipopolysaccharide biosynthesis. This organization is similar to that observed in Rhizobium leguminosarum bv. viciae, where greA is adjacent to lpcC . The conservation of this genetic arrangement across multiple rhizobial species suggests functional significance, potentially indicating coordinated regulation of transcription elongation and cell envelope biosynthesis. The B. japonicum greA gene likely shares sequence homology with other rhizobial greA genes, particularly with R. leguminosarum (which shows 77% identity to S. meliloti greA) .

What expression systems are most effective for producing recombinant GreA from Bradyrhizobium japonicum?

For recombinant production of B. japonicum GreA, Escherichia coli-based expression systems are typically most effective due to their high yield, ease of genetic manipulation, and well-established protocols. Based on research with similar proteins, the following approach is recommended:

Expression system selection:

• pET vector systems (particularly pET28a with an N-terminal His-tag) offer tight regulation and high expression levels

• BL21(DE3) or its derivatives are preferred host strains due to their deficiency in lon and ompT proteases

• For difficult-to-express proteins, specialized strains like Rosetta(DE3) may overcome codon usage bias issues

Expression conditions:

• Induction with 0.1-0.5 mM IPTG at OD₆₀₀ of 0.6-0.8

• Post-induction growth at lower temperatures (16-25°C for 16-18 hours) often improves solubility

• Supplementing growth media with glucose (0.5-1%) can reduce basal expression prior to induction

Alternative eukaryotic expression systems are generally unnecessary for bacterial transcription factors like GreA, as they typically fold correctly in prokaryotic hosts .

What purification strategy yields the highest purity and activity for recombinant GreA protein?

A multi-step purification strategy is recommended for obtaining high-purity, active recombinant GreA protein:

Initial capture:

• Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA resin for His-tagged GreA

  • Lysis buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM DTT

  • Wash buffer: Same as lysis with 20-40 mM imidazole

  • Elution buffer: Same as lysis with 250-300 mM imidazole

Secondary purification:
2. Size Exclusion Chromatography (SEC) using Superdex 75 or similar

• Buffer: 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 1 mM DTT

Optional polishing step:
3. Ion Exchange Chromatography (theoretical pI of GreA should be considered)

• For basic proteins: Cation exchange (SP Sepharose)

• For acidic proteins: Anion exchange (Q Sepharose)

Quality assessment:

• SDS-PAGE: >95% purity

• Western blot: His-tag detection and GreA-specific antibodies if available

• Activity assay: Transcript cleavage assay using stalled transcription complexes

Final storage is optimal in 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 50% glycerol at -80°C, avoiding repeated freeze-thaw cycles .

How can researchers assess the proper folding and activity of purified recombinant GreA?

Multiple complementary approaches should be employed to assess proper folding and activity:

Structural integrity assessment:

• Circular Dichroism (CD) spectroscopy to evaluate secondary structure composition

• Thermal shift assays to determine protein stability and proper folding

• Limited proteolysis to assess compact, folded domains resistant to proteolytic cleavage

• Dynamic Light Scattering (DLS) to evaluate homogeneity and absence of aggregation

Functional activity assays:

• In vitro transcript cleavage assay using:

  • Purified RNA polymerase from B. japonicum or E. coli

  • Template DNA containing a promoter and sequence prone to pausing

  • Visualization of cleaved RNA products by gel electrophoresis

• Binding assays to measure GreA-RNA polymerase interactions:

  • Surface Plasmon Resonance (SPR)

  • Fluorescence Anisotropy with labeled GreA or polymerase components

• Complementation assays in GreA-deficient bacterial strains to assess functionality in vivo

The most definitive evidence of proper folding is demonstration of expected enzymatic activity, which for GreA is the ability to stimulate the transcript cleavage activity of RNA polymerase .

How does GreA from Bradyrhizobium japonicum compare functionally to homologs from other bacterial species?

Comparative analysis of GreA across bacterial species reveals both conserved features and species-specific differences:

Conserved features:

Differences in Bradyrhizobium and other rhizobia:

• Sequence alignment with R. leguminosarum shows approximately 77% identity, indicating significant conservation but with potential functional adaptations

• Genomic context differs among species, with rhizobia commonly having greA positioned near LPS biosynthesis genes, suggesting potential specialized roles in symbiosis

Functional implications:

• B. japonicum GreA may have evolved specific interaction parameters with its cognate RNA polymerase

• The protein might have acquired specialized roles related to symbiotic association with host plants

• Differential regulation of greA expression could reflect adaptation to the slower growth and metabolism characteristic of Bradyrhizobium species (generation times of 9.4-15.7 hours compared to minutes or few hours for E. coli)

A comprehensive understanding requires direct experimental comparison of purified GreA proteins from multiple species in standardized activity assays.

What role might GreA play in the symbiotic relationship between Bradyrhizobium japonicum and legume hosts?

GreA likely plays several critical roles in the complex symbiotic relationship between B. japonicum and legume hosts:

Regulation of symbiosis-specific gene expression:

• Transcription of nod, nif, and fix genes requires precise regulation during establishment of symbiosis

• GreA could help RNA polymerase navigate through GC-rich sequences or complex secondary structures in symbiosis-related genes

Connection to LPS biosynthesis:

• The genetic linkage between greA and LPS biosynthesis genes (as observed in related rhizobia) suggests coordinated regulation

• LPS is crucial for establishing successful plant-microbe interactions and avoiding host defense responses

• GreA may ensure proper expression of LPS synthesis genes during critical phases of nodule development

Stress adaptation during nodule formation:

• The microaerobic, acidic environment of developing nodules imposes transcriptional challenges

• GreA-mediated resolution of paused transcription complexes might be essential for adaptation to these conditions

Metabolic adaptation:

• Transition from free-living to symbiotic lifestyle requires metabolic reprogramming

• GreA could facilitate the required transcriptional shifts during this transition

Research examining differential expression of greA during various stages of symbiosis would provide valuable insights into its symbiotic relevance.

What methods are most effective for studying GreA-RNA polymerase interactions in Bradyrhizobium japonicum?

Several complementary approaches can effectively characterize GreA-RNA polymerase interactions:

In vitro biochemical methods:

• Pull-down assays using His-tagged GreA to capture interacting RNAP subunits

• Surface Plasmon Resonance (SPR) to determine binding kinetics and affinity constants

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

• Single-molecule FRET to observe real-time dynamics of GreA-RNAP interactions during transcription

Structural biology approaches:

• Cryo-electron microscopy of GreA-RNAP complexes at different functional states

• X-ray crystallography of co-crystals containing GreA and RNAP components

• NMR spectroscopy for mapping interaction sites using chemical shift perturbations

In vivo approaches:

• Bacterial two-hybrid systems adapted for B. japonicum

• ChIP-seq to identify genomic regions where GreA associates with transcribing RNAP

• RNA-seq comparing wild-type and greA mutant strains to identify genes most affected by GreA activity

A combination of these methods would provide comprehensive insights into both the physical nature of the interactions and their functional consequences in B. japonicum.

How does nutrient availability affect greA expression in Bradyrhizobium japonicum, and what are the implications for transcriptional regulation?

Nutrient availability significantly impacts B. japonicum metabolism and likely influences greA expression:

Response to varying nutrient conditions:

• B. japonicum strains exhibit distinct growth responses to increasing yeast extract (YE) concentrations, with optimal growth at 1.5-2.0 g/L and growth inhibition at 5 g/L

• These growth patterns suggest sophisticated transcriptional regulation mechanisms that may involve GreA

Potential regulatory mechanisms:

• Nutrient-sensing systems likely modulate greA expression to adjust transcriptional elongation efficiency

• Under nutrient limitation, increased GreA activity could help maintain expression of essential genes by resolving transcriptional pauses caused by low NTP concentrations

• In nutrient-rich conditions, greA regulation might shift to optimize expression of metabolic pathways

Experimental approach to study nutrient effects:

• qRT-PCR analysis of greA expression under varying YE concentrations (0.5-5.0 g/L)

• Western blot quantification of GreA protein levels under different nutrient conditions

• ChIP-seq analysis to map GreA occupancy across the genome under varying nutrient conditions

• Transcriptome analysis of wild-type vs. greA mutant strains under different nutrient regimes

Research implications:

• Understanding greA regulation in response to nutrients could provide insights into B. japonicum's adaptation to the changing nutrient environment during nodule development

• This knowledge could inform strategies to optimize symbiotic performance in agricultural settings

What are the challenges in designing experiments to study the role of GreA in Bradyrhizobium stress responses?

Studying GreA's role in stress responses presents several methodological challenges:

Experimental design challenges:

• Slow growth kinetics: With generation times of 9.4-18.8 hours , experiments require extended timeframes, complicating acute stress response studies

• Genetic manipulation difficulties: Bradyrhizobium's large genome (~9 Mb) and slow growth make genetic modifications time-consuming

• Multiple stress response pathways: Distinguishing GreA-specific effects from other stress response mechanisms requires careful controls

• Environmental sensitivity: B. japonicum exhibits significant experimental variability , necessitating rigorous replication

Methodological considerations and solutions:

Stress conditions relevant to Bradyrhizobium ecology:

• Acidic pH (simulating rhizosphere conditions)

• Microaerobic environments (nodule-like conditions)

• Oxidative stress (host defense response)

• Osmotic stress (soil moisture fluctuations)

• Thermal stress (soil temperature variations)

How might advanced structural biology techniques reveal the mechanism of GreA function in Bradyrhizobium japonicum?

Advanced structural biology techniques can provide critical insights into GreA function at the molecular level:

Cryo-electron microscopy (Cryo-EM):

• Enables visualization of GreA-RNA polymerase complexes in different functional states

• Can capture conformational changes during transcript cleavage

• Resolution now approaching 2-3Å allows visualization of catalytic residues and water molecules

• Sample preparation challenges: Ensuring homogeneity of B. japonicum RNAP-GreA complexes

X-ray crystallography:

• Provides atomic-resolution structures of GreA alone or in complex with RNAP fragments

• Reveals precise details of interaction interfaces and catalytic residues

• Crystallization challenges: Obtaining diffraction-quality crystals of flexible transcription complexes

Integrative structural biology approaches:

• Combining lower-resolution Cryo-EM data with high-resolution crystal structures of components

• Molecular dynamics simulations to model conformational changes during GreA function

• Hydrogen-deuterium exchange mass spectrometry to map protein-protein interaction surfaces

• Cross-linking mass spectrometry (XL-MS) to identify interacting regions

Structural insights likely to be revealed:

• Conformational changes in RNA polymerase induced by GreA binding

• Molecular basis for species-specific differences in GreA function

• Structural consequences of mutations in conserved GreA motifs (PS00829 and PS00830)

• Dynamic rearrangements during transcript cleavage and elongation restart

These structural studies would significantly advance understanding of transcription regulation in Bradyrhizobium and potentially reveal unique adaptations related to its symbiotic lifestyle.

Why might recombinant Bradyrhizobium japonicum GreA show low activity in transcription elongation assays?

Several factors can contribute to low activity of recombinant GreA in functional assays:

Protein-related factors:

• Improper folding: The expression conditions may have resulted in misfolded protein

  • Solution: Try lower induction temperatures (16°C) and slower expression

• Missing post-translational modifications: B. japonicum may modify GreA in ways not replicated in E. coli

  • Solution: Consider expression in related α-proteobacteria hosts

• N- or C-terminal tags interfering with function:

  • Solution: Test constructs with removable tags or different tag positions

Assay-related factors:

• Incompatibility with RNA polymerase source: B. japonicum GreA may have evolved specificity for its cognate RNAP

  • Solution: Use B. japonicum RNAP instead of E. coli RNAP in assays

• Suboptimal buffer conditions:

  • Solution: Systematically optimize salt concentration, pH, and divalent cation concentrations

• Template sequence incompatibility: The DNA template may lack appropriate pause sites

  • Solution: Use templates derived from B. japonicum genes with known regulatory pauses

Experimental validation approaches:

• Circular dichroism to confirm secondary structure integrity

• Size exclusion chromatography to verify monodispersity

• Limited proteolysis to assess folding quality

• Activity comparison with GreA from E. coli or other well-characterized species as positive controls

Systematic troubleshooting of these factors should help identify and address the source of low activity.

What strategies can overcome difficulties in creating stable Bradyrhizobium japonicum greA mutants?

Creating stable greA mutants in B. japonicum presents challenges due to its slow growth and potential essentiality of the gene. The following strategies can help overcome these difficulties:

Genetic manipulation approaches:

• Conditional knockout systems:

  • Tetracycline-inducible expression systems to control greA levels

  • Temperature-sensitive plasmids for controlled gene disruption

• Partial activity mutants:

  • Point mutations in catalytic residues rather than complete gene deletion

  • Domain truncations that preserve some functionality

• CRISPR-Cas9 based approaches:

  • Direct editing of the chromosome without antibiotic markers

  • Multiplex editing to create compensatory mutations if needed

Cultivation considerations:

• Optimized recovery media:

  • YEM medium with 1.5 g/L yeast extract (optimal concentration)

  • Supplemented with osmoprotectants during recovery phase

• Extended recovery periods:

  • Account for the slow growth rate (9.4-18.8 hours generation time)

  • Use microcolony visualization techniques for early detection of transformants

Screening and verification protocols:

• PCR-based screening optimized for high GC content

• RT-qPCR to confirm reduced/eliminated greA expression

• Phenotypic analyses focusing on growth rate and stress responses

• Whole genome sequencing to confirm mutation and detect any compensatory mutations

For essential genes, creating depletion strains rather than complete knockouts may be necessary, allowing for controlled study of GreA function while maintaining viability.

How can researchers differentiate between direct and indirect effects of GreA on gene expression patterns in Bradyrhizobium japonicum?

Differentiating direct from indirect effects of GreA on gene expression requires a multi-layered experimental approach:

Integrated experimental strategy:

• Direct binding and occupancy studies:

  • ChIP-seq to map genome-wide GreA binding sites

  • NET-seq (nascent elongating transcript sequencing) to identify transcriptional pause sites affected by GreA

  • GRO-seq (global run-on sequencing) to measure active transcription in WT vs. greA mutants

• Temporal resolution experiments:

  • Time-course RNA-seq following GreA depletion in conditional mutants

  • Metabolic labeling of newly synthesized RNA to distinguish primary from secondary effects

  • Ribosome profiling to determine translation effects downstream of transcriptional changes

• Perturbation analysis:

  • Testing effects of specific inhibitors of GreA activity

  • Creating point mutations in GreA that affect specific functions

  • Complementation studies with heterologous GreA proteins with known functional differences

Data integration framework:

Statistical approaches:

• Principal component analysis to identify patterns in gene expression changes

• Network analysis to identify regulatory cascades downstream of direct GreA targets

• Bayesian modeling to infer causal relationships between transcriptional events

This integrated approach allows confident classification of gene expression changes as direct consequences of GreA activity versus downstream regulatory effects.

What genomic approaches could reveal novel insights about GreA function in the Bradyrhizobium genus?

Several cutting-edge genomic approaches could significantly advance understanding of GreA function in Bradyrhizobium:

Comparative genomics approaches:

• Pan-genome analysis across multiple Bradyrhizobium species to identify:

  • Conservation patterns in greA sequence and regulatory regions

  • Co-evolution with RNA polymerase components

  • Variation in genomic context of greA among species with different host ranges

• Phylogenomic analysis to correlate GreA sequence variations with:

  • Host specificity

  • Geographical distribution

  • Symbiotic effectiveness

Functional genomics strategies:

• Genome-wide CRISPR interference (CRISPRi) to identify genetic interactions with greA

• Transposon sequencing (Tn-seq) under various stress conditions in wild-type vs. greA mutant backgrounds

• RNA structurome analysis to identify RNA secondary structures affected by GreA activity

• Global proteomics to assess post-transcriptional effects of GreA perturbation

Integrative multi-omics frameworks:

• Correlation of transcriptomic, proteomic, and metabolomic data in response to greA manipulation

• Network modeling to position GreA within the broader regulatory architecture of Bradyrhizobium

• Machine learning approaches to identify subtle patterns in gene expression associated with GreA activity

These genomic approaches could reveal how GreA function has been tailored through evolution to support the unique ecological niche and symbiotic lifestyle of Bradyrhizobium species.

How might knowledge of GreA function be applied to enhance Bradyrhizobium japonicum as a bioinoculant?

Understanding GreA function could lead to several applications for improving B. japonicum as a bioinoculant:

Enhanced stress tolerance engineering:

• If GreA is confirmed to play a key role in stress response, overexpression could enhance survival of bioinoculants in challenging field conditions

• Targeted modifications of GreA to optimize transcriptional response to specific agricultural stresses

Improved symbiotic performance:

• Engineering GreA expression to enhance transcription of key symbiosis genes

• Modifying GreA to optimize expression under the microaerobic conditions found in nodules

• Creating GreA variants that improve coordination between nitrogen fixation and plant growth promotion traits

Biocontrol enhancement:

• Given B. japonicum's demonstrated biocontrol potential (reducing disease severity by 81.25%) , optimizing GreA function could enhance expression of antimicrobial compounds

• Coordinating expression of both plant growth-promoting and biocontrol traits through GreA-mediated transcriptional regulation

Field application considerations:

Research combining laboratory optimization with field trials under diverse environmental conditions would be essential to realize these potential applications.

What emerging technologies might advance our understanding of the temporal dynamics of GreA function during Bradyrhizobium-legume symbiosis?

Several emerging technologies offer promising approaches to study the temporal dynamics of GreA function during symbiosis:

In situ visualization technologies:

• Time-lapse microscopy with fluorescent reporters:

  • GreA-fluorescent protein fusions to track localization during nodule development

  • Dual-color imaging to simultaneously monitor GreA and RNA polymerase

  • Microfluidic devices to observe living bacteria during early infection events

• Advanced tissue imaging techniques:

  • Expansion microscopy of nodule sections to visualize GreA distribution at subcellular resolution

  • Light sheet microscopy for 3D imaging of developing nodules with minimal photodamage

  • Super-resolution microscopy to resolve GreA-RNAP interactions within bacteroids

Temporal transcriptomics approaches:

• Single-cell RNA-seq of bacteria at different stages of symbiosis

• Spatial transcriptomics to map gene expression patterns across nodule zones

• TIME-seq (transient induction measurement by RNA sequencing) to capture rapid transcriptional responses

• SLAM-seq for metabolic labeling of newly synthesized RNA during symbiotic transitions

Biosensor technologies:

• FRET-based sensors to detect GreA activity in real-time

• Optogenetic control of GreA to precisely manipulate its activity during specific symbiotic stages

• Nanobody-based probes for tracking GreA interactions in living cells

Integration with host plant systems:

• Multi-organism transcriptomics to correlate GreA activity with host developmental transitions

• Metabolic flux analysis to link transcriptional regulation to symbiotic metabolism

• Signalome analysis to uncover how plant signals might modulate GreA function

These technologies would provide unprecedented insights into when, where, and how GreA functions during the complex developmental progression of Bradyrhizobium-legume symbiosis.