Recombinant Bradyrhizobium japonicum Chaperone protein DnaK (dnaK), partial

What is the role of DnaK chaperone protein in Bradyrhizobium japonicum?

DnaK is the bacterial homolog of Hsp70 chaperone proteins that plays a critical role in protein homeostasis in B. japonicum. Its primary functions include assisting in proper protein folding, preventing protein aggregation during cellular stress, and facilitating protein transport across membranes. In B. japonicum specifically, DnaK is essential for supporting cellular functions under various stress conditions including heat shock and oxidative stress. The chaperone also plays a crucial role in supporting symbiotic interactions with host legumes by ensuring proper folding of proteins involved in nodulation and nitrogen fixation processes .

How does DnaK interact with other chaperone systems in B. japonicum?

DnaK functions as part of a complex chaperone network in B. japonicum. Research has demonstrated that DnaK directly interacts and collaborates with Hsp90 to reactivate substrate proteins. This interaction has been confirmed through site-directed mutagenesis studies that impaired DnaK binding to Hsp90, resulting in defective chaperone activities. The collaboration between these two major chaperone systems is essential for supporting client protein folding and maintaining protein homeostasis in vivo . This bacterial chaperone collaboration appears to have served as an evolutionary foundation for more complex eukaryotic chaperone systems.

What are the recommended protocols for cloning the B. japonicum dnaK gene?

For cloning the B. japonicum dnaK gene, researchers should follow these methodological steps:

• Genomic DNA extraction: Extract high-quality genomic DNA from B. japonicum using specialized bacterial DNA extraction kits or protocols that account for the Gram-negative nature of the organism.

• PCR amplification: Design specific primers that flank the dnaK coding sequence based on the published B. japonicum genome. Include appropriate restriction sites to facilitate subsequent cloning steps.

• Vector selection: Choose an expression vector compatible with either E. coli or Bradyrhizobium depending on your experimental goals. For structural studies, vectors with histidine or other affinity tags are recommended.

• Restriction digestion and ligation: Digest both the PCR product and vector with appropriate restriction enzymes followed by ligation using T4 DNA ligase.

• Transformation and verification: Transform the ligated products into appropriate host cells, select transformants on antibiotic-containing media, and verify the recombinant clones by colony PCR, restriction analysis, and sequencing .

The techniques used to identify and clone genes from B. japonicum are well-established in molecular biology literature and can be adapted following protocols similar to those used for the nodulation genes described in previous studies .

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

Several expression systems have been successfully used for recombinant DnaK production, each with specific advantages:

E. coli expression systems:

• BL21(DE3) strain with pET vectors is most commonly used due to high protein yields

• Arctic Express strains can be beneficial as they co-express cold-adapted chaperones that aid in proper folding

• Use of T7 promoter systems with IPTG induction allows controlled expression

Native Bradyrhizobium expression:

• Modified Bradyrhizobium strains can be created through electroporation of recombinant plasmids

• Selection of transformed strains can be achieved using antibiotic resistance markers

For optimal protein production, cultivation temperature should be lowered to 16-20°C after induction to minimize aggregation. Fusion tags like 6xHis or GST facilitate purification and can enhance solubility. Expression levels should be monitored through SDS-PAGE and Western blotting using antibodies against DnaK or the affinity tag.

What purification methods yield the highest activity for recombinant DnaK protein?

To obtain high-activity recombinant DnaK protein, a multi-step purification strategy is recommended:

• Affinity chromatography: If using His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin provides good initial purification. For GST-tagged constructs, glutathione sepharose is effective.

• Ion exchange chromatography: Apply the eluted protein from affinity purification to an ion exchange column (typically Q-sepharose) to remove remaining contaminants.

• Size exclusion chromatography: As a final polishing step, gel filtration separates DnaK from aggregates and degradation products.

• ATP treatment: Include ATP washing steps during purification to release substrate proteins that may be bound to DnaK, improving homogeneity.

• Quality control: Assess protein purity by SDS-PAGE (>95% purity) and verify activity through ATPase assays and substrate binding assays.

Maintaining buffer conditions that include 5-10% glycerol and 1-5 mM DTT throughout purification helps preserve protein stability and activity. For highest structural integrity, purification should be performed at 4°C with protease inhibitors present in all buffers.

How can I assess the chaperone activity of recombinant B. japonicum DnaK in vitro?

The chaperone activity of recombinant B. japonicum DnaK can be assessed through multiple complementary assays:

Protein refolding assays:

• Use denatured luciferase or citrate synthase as model substrates

• Monitor the recovery of enzymatic activity over time in the presence of DnaK, DnaJ co-chaperone, and GrpE nucleotide exchange factor

• Compare refolding rates and final yield of active protein with and without DnaK

Protein aggregation prevention assays:

• Heat-denature model proteins like malate dehydrogenase at 43°C

• Measure light scattering at 320-360 nm to quantify aggregation

• Compare aggregation kinetics in the presence and absence of DnaK

ATP hydrolysis assays:

• Measure ATPase activity using colorimetric assays for phosphate release

• Compare basal ATPase activity with substrate-stimulated activity

• Assess the effect of co-chaperones on ATPase rates

Substrate binding assays:

• Use fluorescence anisotropy with labeled peptide substrates

• Determine binding affinity through titration experiments

• Compare binding characteristics with other bacterial DnaK proteins

For meaningful results, these assays should include appropriate controls including commercially available DnaK from E. coli and negative controls without chaperone addition.

What methodologies can reveal the interaction between DnaK and Hsp90 in B. japonicum?

To characterize the interaction between DnaK and Hsp90 in B. japonicum, researchers can employ the following methodologies:

Co-immunoprecipitation (Co-IP):

• Use antibodies against DnaK or Hsp90 to precipitate protein complexes

• Detect interacting partners via Western blot analysis

• Include appropriate controls to confirm specificity of interactions

Pull-down assays:

• Express tagged versions of DnaK or Hsp90 (His, GST)

• Capture complexes using affinity resins

• Identify interacting partners through mass spectrometry or immunoblotting

Surface plasmon resonance (SPR) or bio-layer interferometry (BLI):

• Immobilize purified DnaK or Hsp90 on sensor chips

• Measure real-time association and dissociation kinetics

• Determine binding affinity constants and binding stoichiometry

Site-directed mutagenesis:

• Create point mutations in potential interaction sites based on structural data

• Test the effect of mutations on binding and functional cooperation

• This approach has confirmed that mutations in Hsp90 strongly diminish binding with DnaK

Fluorescence resonance energy transfer (FRET):

• Label DnaK and Hsp90 with appropriate fluorophore pairs

• Measure energy transfer as evidence of protein-protein proximity

• Use in cellular contexts to confirm interactions in vivo

These complementary approaches provide robust evidence of physical interactions and enable mapping of the specific domains involved in DnaK-Hsp90 binding.

How does temperature stress affect DnaK function in B. japonicum?

Temperature stress significantly impacts DnaK function in B. japonicum, with distinct responses observed at different temperature ranges:

Heat shock response (elevated temperatures):

• Increased expression of dnaK gene through sigma-32-dependent transcription

• Enhanced ATPase activity and substrate binding capabilities

• Shift in DnaK localization toward areas of protein aggregation

• Critical role in preventing heat-induced protein aggregation and cellular damage

Studies have demonstrated that Hsp90 mutants with diminished DnaK binding were defective in supporting growth under heat stress conditions in bacteria, highlighting the essential nature of the DnaK-Hsp90 collaboration during temperature stress .

Cold shock response (reduced temperatures):

• Altered substrate specificity profile

• Modified interaction dynamics with co-chaperones

• Potential role in maintaining membrane fluidity through interaction with membrane proteins

Methodological approaches for investigation:

• Growth curve analysis at various temperatures for wild-type and dnaK mutant strains

• Differential gene expression analysis using RNA-seq or qRT-PCR

• Protein aggregation assays comparing wild-type and mutant strains

• Proteomic analysis to identify temperature-dependent DnaK substrates

Temperature adaptation is particularly relevant for B. japonicum as soil bacteria experience significant temperature fluctuations in their natural environment, making DnaK function essential for ecological fitness.

How does DnaK contribute to the nodulation process in B. japonicum-legume symbiosis?

DnaK plays several crucial roles in facilitating successful nodulation during B. japonicum-legume symbiosis:

Proper folding of nodulation proteins:

• Ensures correct folding of NodD transcriptional regulators that respond to plant flavonoids

• Assists in the proper assembly of the nodulation signaling cascade

• Maintains functional integrity of nodABCDIJL gene products essential for nodule formation

Stress protection during infection process:

• Protects bacterial proteins from oxidative stress generated during plant immune response

• Maintains protein homeostasis during pH changes encountered during infection thread formation

• Provides thermal tolerance as nodule temperature may differ from soil temperature

Support for toxin production and virulence factors:

• Similar to how chaperones support biosynthesis of the colibactin toxin in pathogenic E. coli, DnaK likely supports production of molecules involved in symbiotic communication

Methodological approaches for investigation:

• Creation of conditional dnaK mutants to evaluate nodulation efficiency

• Microscopy to track bacterial survival during infection process

• Transcriptome/proteome analysis of wild-type vs. dnaK-deficient strains during nodulation

• Measurement of nitrogen fixation rates to assess functional symbiosis establishment

Research confirms that chaperone activity is essential for proper client protein folding during symbiotic processes, suggesting DnaK's involvement in multiple stages of nodule formation and function.

What experimental approaches can determine DnaK's role in nitrogen fixation efficiency?

To investigate DnaK's contribution to nitrogen fixation efficiency in B. japonicum, researchers can employ these experimental approaches:

Genetic manipulation strategies:

• Generate conditional dnaK mutants using temperature-sensitive alleles or inducible antisense RNA

• Create point mutations in DnaK's substrate binding domain or ATPase domain

• Complement dnaK-deficient strains with wild-type or mutant variants

Physiological and biochemical assays:

• Acetylene reduction assay to quantify nitrogenase activity in nodules formed by wild-type vs. mutant strains

• 15N isotope incorporation studies to measure actual nitrogen fixation rates

• Leghemoglobin content analysis as an indicator of nodule oxygen regulation

• ATP/ADP ratio determination in bacteroids as an energy status indicator

Molecular and cellular analyses:

• Immunolocalization of DnaK during different stages of nodule development

• Co-immunoprecipitation to identify DnaK interactions with nitrogenase components

• Proteomic analysis to identify changes in the nodule proteome in dnaK mutants

• Transcriptome analysis to assess expression changes in nitrogen fixation genes

Plant-based phenotypic measurements:

• Nodule number, size, and morphology comparisons

• Plant biomass and nitrogen content determination

• Root and shoot growth measurements under nitrogen-limited conditions

• Chlorophyll content as an indicator of nitrogen status

These multi-faceted approaches can establish causal relationships between DnaK function and nitrogen fixation efficiency, providing insights into the mechanistic basis of this important symbiotic process.

How can I study the interactions between host plant stress responses and bacterial DnaK function?

To investigate the interplay between host plant stress responses and bacterial DnaK function, implement these research strategies:

Co-cultivation experiments under stress conditions:

• Expose plant-bacteria partnerships to heat, cold, drought, or salinity stress

• Compare nodulation efficiency and nitrogen fixation rates between wild-type and dnaK-modified strains

• Assess bacterial survival rates within nodules under various stress conditions

Multi-omics approaches:

• Conduct RNA-seq on both partners (plant roots and bacteroids) under stress conditions

• Perform comparative proteomics to identify differentially expressed proteins

• Use metabolomics to detect stress-related metabolites exchanged between partners

• Integrate data using bioinformatics to identify key pathways and interactions

Microscopy and imaging techniques:

• Use fluorescent protein fusions to monitor DnaK localization during stress

• Employ electron microscopy to examine ultrastructural changes in bacteroids

• Utilize confocal microscopy with appropriate stains to assess membrane integrity and bacterial viability

Molecular signaling analysis:

• Analyze plant flavonoid production under stress and its impact on bacterial gene expression

• Investigate how bacterial DnaK influences plant hormone signaling pathways

• Study how reactive oxygen species signaling is modified by bacterial chaperone activity

Transgenic approaches:

• Express bacterial DnaK in plant cells to assess cross-kingdom functional conservation

• Modify plant expression of heat shock proteins to examine compensatory effects

• Create reporter systems to visualize stress responses in both partners simultaneously

These methodological approaches provide comprehensive insights into how bacterial DnaK function contributes to symbiotic resilience under environmental stress conditions.

How can site-directed mutagenesis of DnaK reveal functional domains important for B. japonicum symbiosis?

Site-directed mutagenesis of DnaK provides a powerful approach to dissect the functional domains critical for B. japonicum symbiotic interactions:

Key domains for targeted mutagenesis:

• ATP binding domain (N-terminal): Mutations in the ATPase active site (e.g., K70A) can disrupt ATP hydrolysis without affecting substrate binding

• Substrate binding domain (C-terminal): Alterations in the peptide-binding pocket can modify substrate specificity

• Interdomain linker region: Mutations affecting allosteric communication between domains

• Co-chaperone interaction sites: Modifications in regions that interact with DnaJ or GrpE

• Hsp90 interaction interface: Mutations at this interface have been shown to strongly diminish binding with Hsp90

Experimental workflow:

• Design mutations based on sequence alignments and structural data

• Create mutant constructs using standard site-directed mutagenesis techniques

• Express and purify mutant proteins for in vitro characterization

• Reintroduce mutant genes into dnaK-deficient B. japonicum strains

• Assess symbiotic phenotypes through nodulation and nitrogen fixation assays

Functional assays for mutant characterization:

• ATP hydrolysis rates compared to wild-type DnaK

• Substrate binding affinity using model peptides

• Protein refolding efficiency with denatured substrates

• Co-chaperone interaction analysis by pull-down assays

• In vivo complementation of temperature-sensitive growth phenotypes

This approach has successfully identified critical residues in Hsp90 that affect DnaK binding and symbiotic function, suggesting similar strategies would yield valuable insights for DnaK's role in symbiosis .

What are the recommended approaches for studying DnaK's role in evolution of symbiotic capability in Bradyrhizobium species?

To investigate DnaK's evolutionary role in symbiotic capability across Bradyrhizobium species, implement these research approaches:

Comparative genomics and phylogenetics:

• Sequence dnaK genes from multiple Bradyrhizobium strains with varying host ranges

• Construct phylogenetic trees using multiple loci (dnaK, recA, glnII) for robust evolutionary analysis

• Compare dnaK sequence conservation relative to housekeeping and symbiosis genes

• Analyze selection pressures using dN/dS ratios across different functional domains

Ancestral sequence reconstruction:

• Infer ancestral DnaK sequences at key evolutionary nodes

• Synthesize and characterize these ancestral proteins

• Compare chaperone activities between ancestral and modern variants

Horizontal gene transfer analysis:

• Examine if dnaK genes show evidence of horizontal transfer similar to symbiosis island genes

• Use codon usage bias and GC content analysis to identify potential transfer events

• Determine if dnaK evolutionary history parallels symbiosis gene acquisition

Functional complementation experiments:

• Exchange dnaK genes between species with different host specificities

• Assess whether DnaK from one species can restore symbiotic function in another

• Evaluate if DnaK plays a role in host range determination

Correlation with symbiotic phenotypes:

• Map DnaK sequence variations against host range data

• Identify if specific DnaK variants correlate with nodulation capabilities

• Determine whether DnaK sequence changes coincide with evolutionary gains or losses of symbiotic function

These approaches can reveal whether DnaK has played a direct role in the evolution of symbiotic capabilities or has co-evolved with symbiosis genes to support their function.

How can computational approaches predict DnaK substrates relevant to B. japonicum symbiotic function?

Computational approaches offer powerful tools for predicting DnaK substrates relevant to symbiotic function in B. japonicum:

Sequence-based prediction methods:

• Apply established DnaK binding motif algorithms (e.g., limbo, HNDB, ChaperISM)

• Scan B. japonicum symbiosis-related proteins for potential DnaK binding sites

• Prioritize candidates based on binding site accessibility and conservation

Structural bioinformatics approaches:

• Perform molecular docking simulations between DnaK substrate binding domain and candidate proteins

• Use AlphaFold2 or RoseTTAFold to predict structures of symbiotic proteins

• Identify regions with high predicted disorder that may require chaperone assistance for folding

Network analysis methods:

• Construct protein-protein interaction networks focused on symbiotic proteins

• Identify central nodes likely to require chaperone assistance

• Predict functional consequences of disrupted interactions due to misfolding

Integration with -omics data:

• Correlate predicted DnaK substrates with proteins showing altered abundance in dnaK mutants

• Incorporate transcriptomics data to identify co-expressed genes

• Use proteomics data to identify proteins with altered solubility in chaperone mutants

Machine learning approaches:

• Train models using known chaperone substrates from related bacteria

• Apply transfer learning to adapt models to B. japonicum-specific features

• Validate predictions using limited experimental data to refine models

Experiment design for validation:

• Select top predicted substrates for experimental validation

• Express recombinant proteins and test direct binding to DnaK in vitro

• Assess the effect of DnaK on substrate folding and activity

• Confirm biological relevance through in vivo studies

These computational approaches accelerate the identification of key DnaK substrates involved in symbiotic processes, guiding subsequent experimental validation.

What strategies can address poor expression of recombinant B. japonicum DnaK in heterologous systems?

When encountering poor expression of recombinant B. japonicum DnaK, researchers can implement these troubleshooting strategies:

Optimization of expression constructs:

• Codon optimization for the host organism (especially important when expressing in E. coli)

• Testing different affinity tags (His, GST, MBP) and their positions (N- or C-terminal)

• Using stronger or inducible promoters appropriate for the host system

• Including appropriate ribosome binding sites for efficient translation initiation

• Redesigning the construct to remove potential secondary structures in mRNA

Expression host selection:

• Testing multiple E. coli strains (BL21(DE3), Rosetta, Arctic Express, Origami)

• Considering hosts with extra chaperones or rare tRNAs

• Using cell-free expression systems for toxic proteins

• Exploring expression in other bacteria more closely related to Bradyrhizobium

Culture condition optimization:

• Varying induction temperature (typically lower temperatures improve folding)

• Testing different inducer concentrations and induction timing

• Modifying media composition (rich vs. minimal, supplemented with specific nutrients)

• Exploring different aeration conditions and culture densities at induction

Co-expression strategies:

• Co-expressing DnaK with its co-chaperones (DnaJ, GrpE)

• Adding molecular chaperones from E. coli (GroEL/ES, ClpB)

• Including rare tRNA-encoding plasmids

Protein stabilization approaches:

• Adding stabilizing agents to lysis buffer (glycerol, specific ions, mild detergents)

• Including protease inhibitors to prevent degradation

• Testing fusion with solubility-enhancing partners (MBP, SUMO, TrxA)

These approaches have proven successful for expressing problematic proteins from various bacterial species and can be adapted specifically for B. japonicum DnaK expression challenges.

How can I troubleshoot non-functional recombinant DnaK protein in in vitro assays?

When recombinant DnaK shows poor functionality in in vitro assays, consider these troubleshooting approaches:

Protein quality assessment:

• Verify protein integrity by mass spectrometry to confirm full-length product

• Check for proper folding using circular dichroism or intrinsic fluorescence

• Assess aggregate formation through dynamic light scattering

• Confirm ATP binding capacity using fluorescent ATP analogs

• Perform thermal shift assays to evaluate protein stability

Assay condition optimization:

• Systematically vary buffer components (pH, salt concentration, metal ions)

• Test different reducing agents (DTT, β-mercaptoethanol, TCEP)

• Add stabilizing agents like glycerol or specific amino acids

• Optimize protein concentration (too high may lead to aggregation)

• Ensure ATP and Mg2+ concentrations are at optimal ratios

Co-chaperone considerations:

• Include purified co-chaperones (DnaJ and GrpE) in activity assays

• Test co-chaperones from different sources if native ones are unavailable

• Verify the functionality of co-chaperones independently

• Optimize co-chaperone ratios relative to DnaK

Substrate selection:

• Test multiple model substrates (luciferase, rhodanese, citrate synthase)

• Use known DnaK-binding peptides for binding assays

• Consider substrates specific to B. japonicum if standard substrates fail

Technical adjustments:

• Ensure complete removal of affinity tags that might interfere with function

• Check for inhibitory contaminants in protein preparations

• Verify that any equipment used (spectrophotometers, fluorimeters) is properly calibrated

• Include positive controls with commercial chaperones

By systematically addressing these factors, researchers can identify and resolve issues affecting recombinant DnaK functionality in vitro.

What are the common pitfalls when analyzing DnaK-substrate interactions in B. japonicum symbiotic contexts?

When investigating DnaK-substrate interactions in B. japonicum symbiotic contexts, researchers should be aware of these common pitfalls and their solutions:

Technical limitations:

• Pitfall: Cross-reactivity of antibodies between bacterial and plant heat shock proteins
  Solution: Validate antibody specificity using appropriate controls and consider epitope-tagged proteins

• Pitfall: Disruption of native complexes during extraction procedures
  Solution: Use gentle extraction methods, crosslinking approaches, or in vivo proximity labeling

• Pitfall: Background from plant proteins in nodule samples
  Solution: Implement bacteroid purification protocols and use appropriate controls for plant protein contamination

Data interpretation issues:

• Pitfall: Assuming all DnaK-substrate interactions are functionally relevant
  Solution: Validate physiological relevance through genetic approaches and in vivo studies

• Pitfall: Overlooking redundancy in chaperone systems
  Solution: Consider parallel chaperone systems and perform double/triple chaperone knockdowns

• Pitfall: Confusing correlation with causation in omics studies
  Solution: Validate key findings with targeted genetic and biochemical approaches

Methodological improvements:

• Use proximity-dependent biotin identification (BioID) or APEX2 tagging for in vivo interaction studies

• Implement single-cell approaches to account for bacteroid heterogeneity

• Employ advanced microscopy techniques like FRET-FLIM to visualize interactions in intact nodules

• Consider the temporal dynamics of DnaK-substrate interactions during nodule development

What emerging technologies hold promise for advancing DnaK research in B. japonicum?

Several cutting-edge technologies are poised to transform research on DnaK in B. japonicum:

CRISPR-Cas9 genome editing:

• Precise modification of endogenous dnaK gene

• Creation of specific point mutations to analyze domain functions

• Generation of conditional knockdowns for essential genes

• Introduction of reporter fusions at native loci

Cryo-electron microscopy:

• High-resolution structural analysis of DnaK-substrate complexes

• Visualization of DnaK-Hsp90 interactions in different nucleotide states

• Structural insights into conformational changes during chaperone cycle

• Analysis of large chaperone-substrate assemblies difficult to crystallize

Single-molecule techniques:

• FRET-based analysis of DnaK conformational dynamics

• Optical tweezers to study chaperone-mediated protein folding mechanics

• Single-molecule pull-down (SiMPull) for quantitative interaction analysis

• Super-resolution microscopy to visualize DnaK localization in bacteroids

Synthetic biology approaches:

• Designer DnaK variants with altered substrate specificities

• Orthogonal chaperone systems for specific symbiotic functions

• Biosensor development to monitor chaperone activity in vivo

• Engineering stress-responsive circuits involving DnaK

Advanced proteomics techniques:

• Proximity labeling methods (BioID, APEX) to identify transient interactions

• Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Thermal proteome profiling to identify DnaK clients on a proteome-wide scale

• Crosslinking mass spectrometry to capture complex interaction networks

These emerging technologies will enable researchers to address previously intractable questions about DnaK function in symbiotic contexts with unprecedented precision and detail.

How might DnaK research contribute to improving agricultural applications of Bradyrhizobium?

DnaK research has significant potential to enhance agricultural applications of Bradyrhizobium through several translational pathways:

Engineering stress-tolerant inoculants:

• Optimizing DnaK expression levels to enhance bacterial survival in harsh field conditions

• Creating strains with modified DnaK systems for improved heat or drought tolerance

• Developing pre-conditioning treatments that prime the DnaK system before inoculation

• Engineering feedback mechanisms that increase chaperone activity in response to field stressors

Extending host range:

• Understanding how DnaK contributes to host specificity mechanisms

• Engineering DnaK systems that can support nodulation factors for non-traditional hosts

• Developing Bradyrhizobium strains with expanded host range through chaperone optimization

• Creating strains that can effectively compete with indigenous soil bacteria

Improving inoculant production and formulation:

• Optimizing DnaK function to improve bacterial survival during formulation processes

• Developing storage conditions that maintain optimal chaperone function

• Creating preservation methods that protect the proteome through DnaK-focused approaches

• Designing diagnostic tools to assess inoculant quality based on chaperone function

Climate adaptation strategies:

• Developing Bradyrhizobium strains with DnaK systems adapted to future climate scenarios

• Engineering temperature-responsive elements controlling chaperone expression

• Creating strains with enhanced resilience to climate fluctuations through optimized stress response systems

This research direction represents a promising intersection of basic molecular chaperone biology and applied agricultural microbiology with potential global impact.

What unresolved questions about B. japonicum DnaK function would most significantly advance the field?

Several key unresolved questions about B. japonicum DnaK would significantly advance the field if addressed:

Substrate specificity determinants:

• What features distinguish the substrate recognition profile of B. japonicum DnaK from other bacterial DnaK proteins?

• How has DnaK substrate specificity co-evolved with the specific proteins required for symbiosis?

• Can we identify the complete set of symbiosis-specific substrates dependent on DnaK?

Regulatory network integration:

• How is DnaK function integrated with other stress response systems specific to symbiotic contexts?

• What signaling pathways connect plant stress responses to bacterial DnaK function?

• How does the DnaK system communicate with symbiotic development pathways?

Co-chaperone interactions:

• What is the complete set of J-domain proteins that interact with DnaK in B. japonicum?

• How do these co-chaperones direct DnaK activity toward specific symbiotic substrates?

• Are there symbiosis-specific co-chaperones that have evolved for nodulation functions?

Evolutionary perspectives:

• Has the DnaK system undergone specific adaptations in Bradyrhizobium compared to non-symbiotic relatives?

• Did the acquisition of symbiosis islands drive co-evolution of the core chaperone machinery?

• How does DnaK function differ between broadly host-range and narrow host-range strains?

Hsp90-DnaK collaboration:

• What is the structural basis for the essential collaboration between Hsp90 and DnaK in bacteria?

• Which symbiotic proteins specifically require the combined action of both chaperone systems?

• How has this collaboration influenced the evolution of more complex chaperone networks?

Methodological advances needed:

• Development of techniques to study chaperone function in the microaerobic nodule environment

• Methods to visualize and quantify protein folding in vivo during symbiotic interactions

• Approaches to manipulate chaperone function with temporal and spatial precision in symbiotic contexts

Addressing these questions would bridge current knowledge gaps and potentially reveal fundamental principles of chaperone function in bacterial symbiosis.