DnaK E.coli

What is DnaK and what is its function in E. coli?

DnaK is a highly conserved heat shock protein (HSP70) in Escherichia coli that maintains cellular protein homeostasis. As a molecular chaperone, DnaK assists in de novo protein folding, protein disaggregation, protein targeting and translocation through biological membranes, and the remodeling of protein complexes . DnaK was originally identified through mutations that blocked lambda phage DNA replication, and subsequent research has revealed it is essential for E. coli viability . This multifunctional chaperone interacts with more than 700 E. coli proteins, emphasizing its central role in cellular physiology .

How does DnaK expression change under different growth conditions?

DnaK expression is highly regulated and varies significantly with growth conditions, particularly temperature. During normal exponential growth at 37°C, DnaK represents approximately 1% of total cellular proteins (38,500 ± 6,056 molecules per cell in E. coli ATCC 25922) . At lower temperatures (30°C), DnaK levels decrease to about 0.63% of total proteins (18,767 ± 3,044 molecules per cell) . When cells are exposed to heat stress (42°C), DnaK levels increase dramatically to 1.60% of total proteins (103,867 ± 13,735 molecules per cell) . This temperature-dependent expression pattern reflects DnaK's crucial role in the heat shock response.

What is the relationship between DnaK and the E. coli heat shock response?

DnaK is a key component of the E. coli heat shock response, one of the most highly conserved biological responses to environmental changes. The heat shock response induces the synthesis of proteins encoded by the HSP60, HSP70, and HSP90 gene families . During normal growth conditions, DnaK helps maintain proper protein folding, but its expression increases significantly during thermal stress to protect cellular proteins from denaturation and aggregation. The regulation of DnaK synthesis involves complex mechanisms including the heat shock sigma factor (σ32), which controls the transcription of heat shock genes . During stress, DnaK may increase from 1% to up to 13% of total cellular proteins when cells grown at 30°C are exposed to 42°C .

How can researchers accurately quantify DnaK in E. coli samples?

DnaK can be quantified using a competitive enzyme-linked immunosorbent assay (ELISA) method. The procedure involves several key steps:

• Cell culture and harvesting: Grow E. coli to desired phase (typically OD600 = 0.5 for exponential phase)

• Cell lysis: Resuspend cell pellet in 0.1 M NaHPO4 buffer (pH 7.0) and sonicate at 16.8 kilocycles/s for 30 minutes (alternating 1 minute sonication with 1 minute rest on ice)

• Sample preparation: Centrifuge sonicated suspension (13,000 × g, 15 min) and collect supernatant

• ELISA procedure: Use microplates coated with purified DnaK (0.25 μg/ml) and perform competitive ELISA

This method allows detection of DnaK down to approximately 1,500 molecules per cell, enabling precise quantification across various experimental conditions . The ELISA quantification provides more sensitive detection compared to traditional methods such as radioactive markers, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or immunoblotting .

How do DnaK levels vary between different E. coli strains?

Significant variation in DnaK levels has been observed between different E. coli strains, even under identical growth conditions. Research comparing E. coli ATCC 25922 (a clinical isolate) and Epicurian Coli BL21 demonstrated that DnaK concentrations can vary nearly two-fold between strains . When grown in Brain Heart Infusion (BHI) broth, E. coli ATCC 25922 contained 38,500 ± 6,056 DnaK molecules per cell, while Epicurian Coli BL21 contained only 22,000 ± 4,451 molecules per cell . This variation highlights the importance of specifying the exact strain when reporting DnaK quantification data. Interestingly, growth medium appears to have minimal effect on DnaK concentration, as demonstrated by similar values obtained in BHI and Luria broth (38,500 ± 6,056 versus 40,100 ± 6,643 molecules/cell, respectively) .

What are the best methods to study DnaK's ATPase activity?

To study DnaK's ATPase activity, researchers can purify the protein to homogeneity and perform enzymatic assays measuring ATP hydrolysis. The purified DnaK protein exhibits a weak DNA-independent ATPase activity that results in the production of ADP and inorganic phosphate (Pi) . Verification that the observed ATPase activity is encoded by the dnaK+ gene can be achieved by comparing wild-type protein with that from a dnaK756 mutant, which produces an ATPase with altered physical properties .

For interaction studies with other proteins, researchers can assess how DnaK's ATPase activity is modulated. For example, lambda O and P replication proteins interact with DnaK in vitro, with lambda P protein inhibiting DnaK's ATPase activity and lambda O protein stimulating it . These assays provide valuable information about DnaK's functional interactions with client proteins.

How does DnaK influence E. coli central metabolism?

DnaK plays a critical role in E. coli central metabolism, with at least 50% of central metabolism (CM) enzymes interacting with this chaperone . Recent research has revealed that DnaK significantly impacts:

• The hierarchical order of carbon source utilization

• The excretion patterns of main metabolic coproducts

• The distribution of metabolic fluxes throughout central pathways

These findings were established using carbon sources with specific entry points coupled to NMR analyses of real-time carbon assimilation, metabolic coproduct production, and flux rearrangements. The data demonstrate a multilevel interaction between DnaK and central metabolism, suggesting this chaperone is not merely responding to metabolic changes but actively participating in metabolic regulation .

What is the relationship between DnaK levels and bacterial survival after heat stress?

DnaK levels correlate strongly with bacterial survival following heat stress. When E. coli cells are subjected to heat treatments of varying intensity, their survival rates show a significant positive correlation with intracellular DnaK concentration (P < 0.0001), although the relationship is not perfectly linear (R² = 0.6881) . For a given process lethality value, treatments at lower temperatures (50°C) applied for longer times result in both higher DnaK concentrations and higher cell survival rates compared to shorter treatments at higher temperatures (55°C) .

The following table shows DnaK concentrations and cell recovery after heat treatments:

This data indicates that residual DnaK after heating is necessary for cell recovery, and cells actively produce additional DnaK during the recovery process .

How does pre-conditioning with sublethal stress affect DnaK levels and subsequent stress resistance?

Pre-conditioning E. coli with sublethal stress significantly increases DnaK levels and enhances resistance to subsequent lethal stress. Cells subjected to a sublethal heat treatment (55°C, F₇₀¹⁰ = 3 min) followed by 12 hours of recovery accumulate high levels of DnaK (76,786 ± 25,230 molecules/cell) . These cells demonstrate significantly better survival when later exposed to lethal heat treatment (60°C, F₇₀¹⁰ = 5 min) compared to control cells that did not undergo pre-conditioning .

After 24 hours of resuscitation following the lethal treatment, pre-conditioned cells recovered to 7.36 log₁₀ CFU/ml, while non-pre-conditioned cells remained undetectable (<0.7 log₁₀ CFU/ml) . This adaptive response demonstrates that higher intracellular concentrations of DnaK provide enhanced protection against subsequent lethal stress treatments in E. coli, highlighting the protein's critical role in stress adaptation mechanisms.

What are the key cofactors and co-chaperones that interact with DnaK?

DnaK functions as part of a chaperone system that includes several key cofactors and co-chaperones:

• DnaJ (HSP40): Acts as a co-chaperone that delivers substrate proteins to DnaK and stimulates its ATPase activity .

• GrpE: Functions as a nucleotide exchange factor that promotes the release of ADP from DnaK, allowing ATP binding and substrate release .

Together, these proteins form the DnaKJE chaperone system that efficiently assists in protein folding, disaggregation, and other chaperone functions . The coordinated action of these components allows DnaK to cycle through ATP-bound and ADP-bound states, which have different affinities for substrate proteins.

Additionally, DnaK interacts with the lambda phage O and P replication proteins, with lambda P inhibiting DnaK's ATPase activity and lambda O stimulating it . These interactions highlight DnaK's role in phage replication and demonstrate how its activity can be modulated by client proteins.

What genetic suppressors of dnaK mutations have been identified?

Several genetic suppressors of dnaK mutations have been identified, providing insights into DnaK's functional network. A notable example is the dksA gene (dnaK suppressor A), which can suppress the temperature-sensitive growth and filamentation of a dnaK deletion mutant strain when present on a multicopy plasmid .

The dksA gene:

• Maps near the mrcB gene at 3.7 min on the E. coli chromosome

• Encodes a 17,500-Mr polypeptide

• Is not an essential gene, as demonstrated by gene disruption experiments

• Rescues temperature-sensitive growth of dnaK deletion mutants at 40.5°C

• Fully suppresses filamentation at 37°C and partially at 40.5°C

• Does not suppress the inability of dnaK mutant cells to support bacteriophage λ growth

Interestingly, dksA can also suppress the temperature-sensitive growth of a grpE280 mutant strain at 41°C and filamentation at 37°C (but not at 41°C), suggesting it functions in a pathway related to the DnaK-GrpE chaperone system .

How is DnaK post-translationally modified, and what is the functional significance?

DnaK undergoes post-translational modification through phosphorylation both in vitro and in vivo. This modification is believed to result from an autophosphorylation reaction . The phosphorylation state of DnaK may regulate its chaperone activities and interactions with client proteins.

The functional significance of DnaK phosphorylation includes:

• Potential regulation of its ATPase activity

• Modulation of substrate binding and release

• Influence on interactions with co-chaperones and cofactors

• Possible role in signal transduction during stress responses

Further research is needed to fully elucidate the mechanisms and regulatory implications of DnaK phosphorylation, including identification of the specific residues modified and the enzymes involved in regulating this process.

How can DnaK levels be used as a biomarker for stress adaptation in bacteria?

DnaK levels can serve as a valuable biomarker for assessing bacterial stress adaptation and predicting survival. Research indicates that using stress protein measurements to determine cell adaptation and survival may provide more nuanced information than cell counts alone, potentially leading to more efficient heat treatment protocols .

Methodology for using DnaK as a stress biomarker:

• Quantify DnaK using competitive ELISA before and after stress exposure

• Monitor DnaK levels during recovery periods

• Correlate DnaK concentrations with survival rates under subsequent stress conditions

• Use DnaK threshold values to predict bacterial resistance to processing treatments

This approach provides insights into cellular physiological states that may not be apparent from viability measurements alone. Cells with elevated DnaK levels but undetectable growth on standard media may be in a viable but non-culturable state, with the potential to recover under favorable conditions .

What methodological approaches can identify the complete interactome of DnaK in E. coli?

Identifying DnaK's complete interactome requires sophisticated methodological approaches:

• In vivo crosslinking coupled with immunoprecipitation: Stabilize transient protein-protein interactions before cell lysis

• Tandem affinity purification (TAP) tagging: Express DnaK with affinity tags for sequential purification steps

• Mass spectrometry analysis: Identify co-purifying proteins with high sensitivity

• Proximity-based labeling: Use techniques like BioID or APEX to label proteins in close proximity to DnaK in living cells

• Computational predictions and network analysis: Integrate experimental data with bioinformatic predictions

These approaches have revealed that DnaK interacts with over 700 E. coli proteins, including at least 50% of central metabolism enzymes . The comprehensive interactome provides insights into DnaK's diverse cellular functions and regulatory networks.

How do mutations in DnaK affect its role in central metabolism and stress response?

Mutations in DnaK can have profound effects on its functions in central metabolism and stress response. Different mutations may:

• Alter ATPase activity, as demonstrated by the dnaK756 mutation producing an ATPase with modified physical properties

• Disrupt client protein interactions, affecting metabolic enzyme folding and function

• Change the hierarchical order of carbon source utilization and metabolic flux distribution

• Impair heat shock response and thermotolerance

• Affect interactions with co-chaperones like DnaJ and GrpE

Methodological approaches to study these effects include:

• Engineering specific mutations through site-directed mutagenesis

• Complementation studies in dnaK deletion strains

• Metabolic flux analysis using NMR or mass spectrometry

• Protein-protein interaction studies with mutant variants

• In vivo phenotypic analysis under various stress conditions

Understanding how different DnaK mutations affect specific functions provides insights into structure-function relationships and the molecular mechanisms underlying its diverse cellular roles.

What are the emerging techniques for studying DnaK dynamics in living cells?

Several cutting-edge techniques are emerging for studying DnaK dynamics in living cells:

• Fluorescence resonance energy transfer (FRET): Monitoring real-time interactions between DnaK and client proteins or co-chaperones

• Single-molecule tracking: Following individual DnaK molecules in living cells to map their subcellular localization and movement

• Optogenetics: Using light-sensitive domains to control DnaK activity with spatial and temporal precision

• Cryo-electron tomography: Visualizing DnaK-substrate complexes in their native cellular context

• Time-resolved proteomics: Tracking changes in DnaK interactome during stress responses

These approaches provide unprecedented insights into DnaK's dynamic behavior in living cells, including its spatial distribution, temporal regulation, and client protein selection under different physiological conditions.

How can systems biology approaches integrate DnaK function into cellular networks?

Systems biology approaches offer powerful frameworks for integrating DnaK function into comprehensive cellular networks:

• Multi-omics integration: Combining proteomics, metabolomics, and transcriptomics data to map DnaK's influence across cellular systems

• Mathematical modeling: Developing quantitative models of DnaK's role in protein homeostasis and stress response

• Network analysis: Identifying key nodes and regulatory hubs connected to DnaK function

• Genome-scale metabolic models: Incorporating chaperone functions into metabolic network reconstructions

• Machine learning approaches: Predicting DnaK substrates and regulatory interactions from large datasets

These integrative approaches help contextualize DnaK's functions within the broader cellular physiological landscape, revealing emergent properties and system-level effects that may not be apparent from reductionist studies.

What therapeutic applications might emerge from understanding DnaK's role in bacterial stress response?

Understanding DnaK's role in bacterial stress response has potential therapeutic applications:

• Novel antimicrobial targets: Developing compounds that inhibit DnaK function to sensitize bacteria to environmental stresses

• Bioprocess optimization: Engineering strains with modified DnaK expression for enhanced production of biopharmaceuticals or industrial enzymes

• Probiotics development: Creating stress-resistant beneficial bacteria for improved gut colonization

• Vaccine development: Utilizing knowledge of heat shock response to design attenuated live vaccines

• Food safety: Implementing more effective thermal processing protocols based on DnaK dynamics