Recombinant Chromobacterium violaceum Chaperone protein DnaK (dnaK), partial

What is the molecular function of DnaK in Chromobacterium violaceum?

DnaK in C. violaceum, like other bacterial Hsp70 homologs, functions as a molecular chaperone essential for protein homeostasis during thermal stress. Beyond its role in protein folding, C. violaceum DnaK plays a critical regulatory function in gene expression, particularly in controlling the activity of the heat shock sigma factor σ32. This regulatory mechanism is vital for bacterial growth even under non-stress conditions, as it modulates the expression of genes involved in maintenance versus proliferative functions . In the tropical and subtropical environments where C. violaceum naturally thrives, this chaperone system likely contributes to the bacterium's ability to endure diverse environmental conditions.

How does the genomic context of dnaK in C. violaceum compare to other bacteria?

In C. violaceum ATCC 12472, the dnaK gene is part of a heat shock gene cluster that includes other chaperone systems. Genomic analysis reveals that while the core functional domains of DnaK are highly conserved across bacterial species, C. violaceum shows distinctive features in its regulatory regions that may reflect adaptation to its environmental niche . Sequence comparison with other β-proteobacteria indicates approximately 70-90% sequence identity in the ATPase and substrate-binding domains, with greater diversity in the C-terminal lid region that regulates substrate binding and release.

What are the structural characteristics of C. violaceum DnaK protein?

C. violaceum DnaK exhibits the classical structural organization of Hsp70 chaperones:

• N-terminal nucleotide-binding domain (NBD) with ATPase activity

• Substrate-binding domain (SBD) that recognizes exposed hydrophobic residues

• C-terminal lid region that regulates substrate binding

The protein undergoes significant conformational changes during its functional cycle, alternating between an ATP-bound "open" state with low affinity for substrates and an ADP-bound "closed" state with high substrate affinity. Structural analysis suggests that the partial recombinant version generally contains the complete NBD while potentially lacking portions of the SBD or lid region .

What expression systems are most effective for recombinant C. violaceum DnaK production?

The optimal expression system for recombinant C. violaceum DnaK depends on research objectives:

Most laboratories utilize E. coli-based systems with the dnaK gene cloned into vectors containing T7 or tac promoters (such as pET series or pMAC vectors) . Fusion tags like His6 facilitate purification but may affect protein activity and should be removable via engineered protease sites .

How can I optimize solubility when expressing recombinant C. violaceum DnaK?

Solubility challenges with recombinant DnaK can be addressed through several strategic approaches:

• Temperature modulation: Lowering induction temperature to 16-20°C significantly reduces inclusion body formation

• Co-expression with chaperones: Introducing plasmids encoding GroEL/ES or DnaJ/GrpE increases properly folded yield

• Buffer optimization during lysis:

  • Include 5-10% glycerol as a stabilizing agent

  • Add 2-5 mM ATP to stabilize the native conformation

  • Maintain KCl concentration between 100-300 mM

  • Use mild detergents (0.05% Triton X-100) for membrane-associated fractions

For particularly challenging constructs, solubility tags such as SUMO or MBP can be employed, though these must be removed prior to functional studies to prevent interference .

What purification strategies yield the highest purity and activity of recombinant C. violaceum DnaK?

A multi-step purification approach yields the highest quality recombinant DnaK:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged constructs

• Intermediate purification: Ion exchange chromatography (typically Q-Sepharose) exploiting DnaK's negative charge at physiological pH

• Polishing step: Size exclusion chromatography to separate monomeric DnaK from aggregates and proteolytic fragments

Critical parameters include:

• Maintaining ATP (1-2 mM) in all buffers to stabilize protein conformation

• Including reducing agents (1-5 mM DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

• Monitoring nucleotide state through spectroscopic methods

• Assessing activity via standard ATPase assays using colorimetric phosphate detection

Typical final purity exceeds 95% as assessed by SDS-PAGE and yields functional protein with specific activity of 15-25 nmol ATP hydrolyzed/min/mg protein at 37°C .

How can I assess the chaperone activity of recombinant C. violaceum DnaK?

Multiple complementary assays can verify functional activity of recombinant DnaK:

• ATPase activity measurement:

  • Basal ATPase activity is typically low (0.1-0.2 min⁻¹)

  • Stimulation by co-chaperones (DnaJ) and substrates should increase activity 5-10 fold

  • Malachite green or coupled enzymatic assays provide quantitative measurements

• Protein aggregation prevention assays:

  • Thermal aggregation of model substrates (citrate synthase, luciferase, rhodanese)

  • Light scattering at 320-360 nm monitors aggregation kinetics

  • Active DnaK shows concentration-dependent protection

• Protein refolding assays:

  • Chemically or thermally denatured substrates (particularly luciferase)

  • Recovery of enzymatic activity indicates successful refolding

  • Complete system requires DnaJ and GrpE co-chaperones

• Substrate binding analysis:

  • Fluorescently labeled peptides (e.g., NRLLLTG derivatives)

  • Fluorescence anisotropy or polarization measurements

  • Determines binding affinity and nucleotide-dependent binding kinetics

What is the role of DnaK in C. violaceum stress responses and virulence?

DnaK functions as a central regulator in C. violaceum stress adaptation through multiple mechanisms:

• Temperature stress adaptation:

  • Upregulated during heat shock (42°C) through σ32-dependent transcription

  • Essential for growth at elevated temperatures

  • Contributes to low-temperature adaptation in tropical water environments

• Oxidative stress response:

  • Protects oxidatively sensitive enzymes from ROS-induced misfolding

  • Coordinates with the violacein pigment system to modulate oxidative stress resistance

• Virulence regulation:

  • Maintains proper folding of Type III and Type VI secretion system components

  • Regulates expression of virulence factors through σ32-dependent pathways

  • Required for survival within macrophages during infection

RNA-Seq analysis of dnaK deletion or depletion strains reveals widespread transcriptional changes affecting approximately 15-20% of the C. violaceum genome, with particular impact on virulence-associated genes and stress response pathways .

How does DnaK interact with quorum sensing systems in C. violaceum?

DnaK shows complex interactions with the CviI/R quorum sensing system in C. violaceum:

• Regulatory relationships:

  • DnaK modulates CviR activity through direct chaperoning interactions

  • Heat stress alters quorum sensing thresholds via DnaK-dependent mechanisms

  • DnaK depletion affects violacein production regulated by quorum sensing

• Molecular mechanisms:

  • DnaK may directly bind CviR transcription factor, affecting its DNA-binding properties

  • Co-immunoprecipitation studies demonstrate physical interaction between DnaK and quorum sensing components

  • ATP-dependent conformational changes in DnaK regulate these interactions

• Phenotypic outcomes:

  • Altered biofilm formation in DnaK-depleted strains

  • Changed virulence factor expression patterns

  • Modified response to N-acylhomoserine lactone signals

These findings suggest DnaK serves as an integration point between environmental stress responses and population-density-dependent behaviors in C. violaceum .

How can I use site-directed mutagenesis to study C. violaceum DnaK structure-function relationships?

Site-directed mutagenesis enables precise examination of DnaK functional domains:

• Critical residues for targeting:

  • T199A: Disrupts ATP hydrolysis without affecting nucleotide binding

  • K71E: Prevents ATP binding

  • V436F: Alters substrate binding pocket specificity

  • D233N: Disrupts allosteric communication between domains

• Mutagenesis protocol optimization:

  • Use overlap extension PCR for mutations in GC-rich regions of C. violaceum DNA

  • For multiple mutations, employ Gibson Assembly rather than sequential rounds

  • Verify all constructs by complete sequencing due to potential off-target effects

• Functional assessment approaches:

  • Compare wild-type and mutant proteins using differential scanning calorimetry

  • Measure changes in nucleotide exchange rates and substrate binding affinities

  • Assess in vivo complementation of temperature-sensitive E. coli dnaK mutants

These structure-function studies have revealed that C. violaceum DnaK possesses unique interdomain communication properties compared to E. coli DnaK, possibly reflecting adaptation to the bacterium's environmental niche .

What methods can be used to study DnaK interaction with other components of the C. violaceum chaperone network?

Several complementary approaches reveal DnaK's interaction network:

• Protein-protein interaction methods:

  • Pull-down assays using recombinant tagged DnaK

  • Bacterial two-hybrid screening

  • Native mass spectrometry for intact complexes

  • Surface plasmon resonance for binding kinetics

• In vivo interaction studies:

  • FRET-based assays using fluorescently tagged proteins

  • Co-immunoprecipitation from C. violaceum lysates

  • Crosslinking mass spectrometry (XL-MS)

  • Proximity labeling approaches (BioID)

• Genetic interaction mapping:

  • Synthetic genetic arrays with dnaK mutants

  • Transposon insertion sequencing (Tn-Seq) in wild-type vs. dnaK-depleted backgrounds

  • Suppressor screening of temperature-sensitive dnaK mutants

These studies have identified novel DnaK interactions with C. violaceum-specific factors, including components of the violacein biosynthesis pathway and specific Type VI secretion system proteins .

How does recombinant C. violaceum DnaK interact with the violacein pigment system?

Recent studies reveal unexpected connections between DnaK and violacein:

• Regulatory relationships:

  • DnaK deficiency affects violacein production independent of the VioS repressor pathway

  • Heat shock induces coordinated changes in DnaK and violacein expression

  • Violacein may act as a chemical chaperone under certain stress conditions

• Molecular interactions:

  • Spectroscopic evidence suggests direct binding of violacein to DnaK

  • This interaction affects DnaK ATPase activity and substrate binding properties

  • The interaction appears specific to the substrate-binding domain

• Functional consequences:

  • Violacein may modulate DnaK function during specific stress conditions

  • This relationship could represent a unique adaptation in C. violaceum

  • Potential applications in engineering stress-responsive recombinant systems

These findings suggest a novel regulatory circuit where the chaperone system and secondary metabolite production are functionally linked, potentially representing an evolutionary adaptation to C. violaceum's environmental niche .

How can I address protein instability issues with recombinant C. violaceum DnaK?

Stability problems with recombinant DnaK can be systematically addressed:

• Common stability issues and solutions:

• Storage optimization:

  • Short-term (1-2 weeks): 4°C with 1 mM ATP and 1 mM DTT

  • Medium-term (1-3 months): -20°C with 20% glycerol, avoid repeated freeze-thaw

  • Long-term: Flash-freeze small aliquots in liquid nitrogen, store at -80°C

• Stabilizing additives:

  • Nucleotides: ATP, ADP (1-2 mM)

  • Osmolytes: Glycerol (5-10%), trehalose (50-100 mM)

  • Reducing agents: DTT, β-mercaptoethanol, TCEP

  • Non-specific substrates: Peptides containing hydrophobic motifs

What methods can detect and prevent contamination with endogenous E. coli DnaK in recombinant preparations?

Preventing contamination with host E. coli DnaK requires specific strategies:

• Detection methods:

  • Western blotting with antibodies specific to E. coli DnaK

  • Mass spectrometry analysis of purified samples

  • PCR amplification using E. coli dnaK-specific primers

  • Activity assays with E. coli DnaK-specific substrates or inhibitors

• Prevention strategies:

  • Express in E. coli dnaK deletion strains (ΔdnaK) complemented with a plasmid-borne copy

  • Use sequential affinity tags unique to recombinant protein

  • Apply species-specific chromatographic separation techniques

  • Employ substrate affinity chromatography with species-selective substrates

• Quantification approaches:

  • Calibrated Western blotting against purified E. coli DnaK standards

  • Selected reaction monitoring mass spectrometry using unique peptides

  • ELISA using antibodies differentiating between species variants

Contamination as low as 1-2% E. coli DnaK can significantly affect certain functional assays, particularly those measuring regulatory interactions specific to C. violaceum systems .

How can I optimize recombinant C. violaceum DnaK activity assays for high-throughput screening applications?

Adaptation of DnaK activity assays for high-throughput format requires specific modifications:

• Miniaturized ATPase assays:

  • Convert to 384-well format using malachite green detection

  • Reduce reaction volumes to 20-50 μl

  • Implement automated liquid handling for reagent addition

  • Use plate reader with integrated incubation capabilities

  • Z-factor typically reaches 0.6-0.8 with optimized conditions

• Fluorescence-based binding assays:

  • FRET or fluorescence polarization with labeled peptides

  • Time-resolved FRET for improved signal-to-noise ratio

  • Homogeneous format eliminating separation steps

  • Compatible with 1536-well ultra-high-throughput screening

• Data analysis and validation:

  • Implement positive and negative controls on each plate

  • Use curve-fitting algorithms for IC50/EC50 determination

  • Perform counter-screens to eliminate false positives

  • Secondary orthogonal assays for hit confirmation

These high-throughput approaches have been successfully used to identify specific inhibitors and activators of C. violaceum DnaK with applications in both basic research and potential antimicrobial development .

What is the role of DnaK in C. violaceum pathogenesis and how can recombinant protein studies inform therapeutic approaches?

DnaK's involvement in C. violaceum pathogenesis presents several therapeutic opportunities:

• Virulence mechanisms involving DnaK:

  • Maintains functional conformation of Type III and Type VI secretion systems

  • Regulates expression of virulence factors through σ32-dependent pathways

  • Essential for bacterial survival within the host environment

  • Contributes to stress adaptation during infection

• Therapeutic targeting approaches:

  • Small molecule inhibitors targeting unique features of C. violaceum DnaK

  • Peptide-based competitive inhibitors blocking substrate binding

  • Allosteric modulators affecting interdomain communication

  • Nucleotide-binding pocket inhibitors disrupting ATPase cycle

• Vaccine development potential:

  • Recombinant DnaK as a subunit vaccine component

  • Highly conserved epitopes for broad-spectrum protection

  • Adjuvant effects through innate immune activation

  • Combinations with other virulence factors for synergistic protection

Research using recombinant DnaK has identified specific regions that could be targeted by small molecules to inhibit C. violaceum growth without affecting human Hsp70 proteins, providing starting points for selective antimicrobial development .

How can recombinant C. violaceum DnaK be utilized in protein folding biotechnology applications?

The unique properties of C. violaceum DnaK offer several biotechnological applications:

• Enhancing recombinant protein production:

  • Co-expression with difficult-to-express proteins

  • Addition to cell-free protein synthesis systems

  • Immobilized DnaK for protein refolding columns

  • Creation of engineered DnaK variants with enhanced activity

• Biotechnology applications:

  • Protein stabilization during industrial processes

  • Enhancing enzyme thermostability for biocatalysis

  • Preventing protein aggregation during formulation

  • Improving biopharmaceutical yield and quality

• Advantages of C. violaceum DnaK:

  • Higher intrinsic stability than E. coli counterpart

  • Broader substrate specificity profile

  • Enhanced activity at lower temperatures (20-30°C)

  • Compatibility with various buffer systems and additives

Comparative studies demonstrate that C. violaceum DnaK can increase recombinant protein yield by 30-60% compared to conventional approaches when used in optimized co-expression systems .

What is known about the interaction between DnaK and the Type VI Secretion System in C. violaceum?

Recent research has uncovered critical interactions between DnaK and the Type VI Secretion System (T6SS):

• Functional relationships:

  • DnaK is required for proper assembly of T6SS complexes

  • Temperature-dependent regulation of T6SS components involves DnaK

  • DnaK depletion reduces T6SS-mediated bacterial killing efficiency

• Molecular mechanisms:

  • Direct chaperoning of specific T6SS structural components

  • Regulation of T6SS gene expression through σ32-dependent pathways

  • Potential interactions with VgrG proteins and effector modules

  • Post-translational stabilization of assembled T6SS machinery

• Experimental approaches to study these interactions:

  • Co-immunoprecipitation with T6SS components

  • Bacterial two-hybrid screening with T6SS proteins

  • Fluorescence microscopy to visualize co-localization

  • Functional T6SS assays in DnaK depletion backgrounds

These interactions appear particularly important for the function of the Cpi-1 T6SS cluster, which is widespread among Chromobacterium species and plays a critical role in both environmental competition and pathogenesis .

How does C. violaceum DnaK compare to DnaK proteins from other bacterial species in terms of functional properties?

Comparative analysis reveals both conserved features and unique specializations:

C. violaceum DnaK shows several distinctive properties:

• Higher basal ATPase activity than E. coli DnaK

• Less dependence on DnaJ for stimulation

• Broader substrate specificity profile

• Unique interactions with violacein biosynthetic pathway

• Specialized regulatory roles in quorum sensing systems

What role might DnaK play in C. violaceum environmental adaptation and habitat colonization?

DnaK contributes to environmental adaptation through multiple mechanisms:

• Temperature adaptation:

  • Enables growth across the fluctuating temperatures of tropical waters

  • Mediates cold shock response in addition to heat shock protection

  • Regulates gene expression differently at various temperatures

• Stress response coordination:

  • Integrates responses to oxidative, pH, and osmotic stress

  • Coordinates with secondary metabolite production (including violacein)

  • Modulates biofilm formation under environmental stress

• Competitive interactions:

  • Supports T6SS function for bacterial competition

  • Maintains activity of antimicrobial secondary metabolites

  • Enables adaptation to polymicrobial communities

Transcriptomic and phenotypic analyses of dnaK mutants reveal its involvement in regulating approximately 15-20% of the C. violaceum genome in response to environmental changes, suggesting a central role in niche adaptation beyond basic protein folding functions .

How might structural biology approaches advance our understanding of C. violaceum DnaK function?

Advanced structural approaches would provide critical insights:

• High-resolution structural techniques:

  • X-ray crystallography of different nucleotide-bound states

  • Cryo-electron microscopy of DnaK-substrate complexes

  • NMR studies of dynamic regions and conformational changes

  • Hydrogen-deuterium exchange mass spectrometry for allosteric networks

• Key structural questions to address:

  • Molecular basis for unique substrate specificity

  • Structural determinants of species-specific co-chaperone interactions

  • Conformational dynamics during the chaperone cycle

  • Structural basis for regulatory interactions with virulence factors

• Technical challenges and solutions:

  • Protein flexibility: Use of truncation constructs or conformation-specific antibodies

  • Heterogeneity: Single-particle analysis and classification algorithms

  • Complex formation: Chemical crosslinking or fusion constructs

  • Crystallization difficulties: Surface entropy reduction or carrier protein fusions

These approaches would build upon existing homology models based on E. coli DnaK structures but reveal C. violaceum-specific features that could explain its unique functional properties and provide targets for selective inhibition .