Hsp33 Controls Elongation Factor-Tu Stability and Allows Escherichia coli Growth in the Absence of the Major DnaK and Trigger Factor Chaperones

Intracellular de novo protein folding is assisted by cellular networks of molecular chaperones. In Escherichia coli, cooperation between the chaperones trigger factor (TF) and DnaK is central to this process. Accordingly, the simultaneous deletion of both chaperone-encoding genes leads to severe growth and protein folding defects. Herein, we took advantage of such defective phenotypes to further elucidate the interactions of chaperone networks in vivo. We show that disruption of the TF/DnaK chaperone pathway is efficiently rescued by overexpression of the redox-regulated chaperone Hsp33. Consistent with this observation, the deletion of hslO, the Hsp33 structural gene, is no longer tolerated in the absence of the TF/DnaK pathway. However, in contrast with other chaperones like GroEL or SecB, suppression by Hsp33 was not attributed to its potential overlapping general chaperone function(s). Instead, we show that overexpressed Hsp33 specifically binds to elongation factor-Tu (EF-Tu) and targets it for degradation by the protease Lon. This synergistic action of Hsp33 and Lon was responsible for the rescue of bacterial growth in the absence of TF and DnaK, by presumably restoring the coupling between translation and the downstream folding capacity of the cell. In support of this hypothesis, we show that overexpression of the stress-responsive toxin HipA, which inhibits EF-Tu, also rescues bacterial growth and protein folding in the absence of TF and DnaK. The relevance for such a convergence of networks of chaperones and proteases acting directly on EF-Tu to modulate the intracellular rate of protein synthesis in response to protein aggregation is discussed.     

Co-translational involvement of the chaperonin GroEL in the folding of newly translated polypeptides

A large fraction of the newly translated polypeptides emerging from the ribosome require certain proteins, the so-called molecular chaperones, to assist in their folding. In Escherichia coli, three major chaperone systems are considered to contribute to the folding of newly synthesized cytosolic polypeptides. Trigger factor (TF), a ribosome-tethered chaperone, and DnaK are known to exhibit overlapping co-translational roles, whereas the cage-shaped GroEL, with the aid of the co-chaperonin, GroES, and ATP, is believed to be implicated in folding only after the polypeptides are released from the ribosome. However, the recent finding that GroEL-GroES overproduction permits the growth of E. coli cells lacking both TF and DnaK raised questions regarding the separate roles of these chaperones. Here, we report the puromycin-sensitive association of GroEL-GroES with translating ribosomes in vivo. Further experiments in vitro, using a reconstituted cell-free translation system, clearly demonstrate that GroEL associates with the translation complex and accomplishes proper folding by encapsulating the newly translated polypeptides in the central cavity formed by GroES. Therefore, we propose that GroEL is a versatile chaperone, which participates in the folding pathway co-translationally and also achieves correct folding post-translationally.     

An Essential Nonredundant Role for Mycobacterial DnaK in Native Protein Folding

Protein chaperones are essential in all domains of life to prevent and resolve protein misfolding during translation and proteotoxic stress. HSP70 family chaperones, including E. coli DnaK, function in stress induced protein refolding and degradation, but are dispensable for cellular viability due to redundant chaperone systems that prevent global nascent peptide insolubility. However, the function of HSP70 chaperones in mycobacteria, a genus that includes multiple human pathogens, has not been examined. We find that mycobacterial DnaK is essential for cell growth and required for native protein folding in Mycobacterium smegmatis. Loss of DnaK is accompanied by proteotoxic collapse characterized by the accumulation of insoluble newly synthesized proteins. DnaK is required for solubility of large multimodular lipid synthases, including the essential lipid synthase FASI, and DnaK loss is accompanied by disruption of membrane structure and increased cell permeability. Trigger Factor is nonessential and has a minor role in native protein folding that is only evident in the absence of DnaK. In unstressed cells, DnaK localizes to multiple, dynamic foci, but relocalizes to focal protein aggregates during stationary phase or upon expression of aggregating peptides. Mycobacterial cells restart cell growth after proteotoxic stress by isolating persistent DnaK containing protein aggregates away from daughter cells. These results reveal unanticipated essential nonredunant roles for mycobacterial DnaK in mycobacteria and indicate that DnaK defines a unique susceptibility point in the mycobacterial proteostasis network.     

Improvement of productivity of active form of glutamate racemase in Escherichia coli by coexpression of folding accessory proteins

Since two classes of folding accessory proteins, molecular chaperones and foldases, prevent the misfolding of newly synthesized polypeptides in the cell, their coexpression could be expected to improve the productivity of soluble and active recombinant proteins. Escherichia coli cytoplasmic glutamate racemase (GluR), which has five cysteine thiol groups and no disulfide bond, was selected as a model enzyme and overexpressed in E. coli. The effects of coexpressing a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active GluR in E. coli were examined. A relatively large amount of active GluR produced by mild induction with 10 μM isopropyl-β-d-thiogalactopyranoside (IPTG). Active GluR productivity was further increased 2.2–2.3-fold by coexpression of GroEL/ES, Trx, or DsbB–DsbD (DsbBD), while it was decreased by coexpression of DnaK–DnaJ–GrpE and TF. These results demonstrate that coexpression of appropriate folding accessory proteins could significantly improve the productivity of active form of proteins in E. coli.     

Functional Differences Between Cyanobacterial DnaK1 Chaperones from the Halophyte Aphanothece halophytica and the Freshwater Species Synechococcus elongatus Expressed in Escherichia coli

DnaK chaperones participate in essential cellular processes including the assistance of the folding, structural maintenance, trafficking, and degradation of proteins, the control of stress responses, and so on. In contrast to the situation found in most other bacterial groups, the cyanobacteria contain multiple dnaK homolog genes whose cellular roles remain ambiguous. We compared in this work the in vivo chaperone capabilities of the DnaK1 members from the halophyte Aphanothece halophytica and the freshwater species Synechococcus elongatus. The corresponding dnaK1 genes were expressed in Escherichia coli, and the abilities of the encoded chaperones to provide for both general and specific functions conducted by E. coli DnaK were analyzed. Synechococcus DnaK1 was far more effective than A. halophytica DnaK1 in replacing E. coli DnaK in all activities tested in vivo, including changes in cell morphology and downregulation of the heat shock response, prevention of the aggregation of misfolded proteins, and restoration of thermotolerance to dnaK-deficient mutants. Thus, regardless of an extensive sequence similarity and comparable in vitro chaperone capabilities, the two cyanobacterial DnaK1 chaperones functionally differed under in vivo conditions. The overall results reinforce the notion that A. halophytica DnaK1 and Synechococcus DnaK1 evolved different substrate specificity since they separated from a common ancestor.     

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.     

A Homolog of Bacillus subtilis Trigger Factor in Listeria monocytogenes Is Involved in Stress Tolerance and Bacterial Virulence

Molecular chaperones play an essential role in the folding of nascent chain polypeptides, as well as in the refolding and degradation of misfolded or aggregated proteins. They also assist in protein translocation and participate in stress functions. We identified a gene, designated tig, encoding a protein homologous to trigger factor (TF), a cytosolic ribosome-associated chaperone, in the genome of Listeria monocytogenes. We constructed a chromosomal Δtig deletion and evaluated the impact of the mutation on bacterial growth in broth under various stress conditions and on pathogenesis. The Δtig deletion did not affect cell viability but impaired survival in the presence of heat and ethanol stresses. We also identified the ffh gene, encoding a protein homologous to the SRP54 eukaryotic component of the signal recognition particle. However, a Δffh deletion was not tolerated, suggesting that Ffh is essential, as it is in Bacillus subtilis and Escherichia coli. Thus, although dispensable for growth, TF is involved in the stress response of L. monocytogenes. The Δtig mutant showed no or very modest intracellular survival defects in eukaryotic cells. However, in vivo it showed a reduced capacity to persist in the spleens and livers of infected mice, revealing that TF has a role in the pathogenicity of L. monocytogenes.     

Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H^D. Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H^D. We find that K/J/E slows down RNase H^D''s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H^D with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.     

Trigger factor assists the refolding of heterodimeric but not monomeric luciferases

The refolding of thermally inactivated protein by ATP-independent trigger factor (TF) and ATP-dependent DnaKJE chaperones was comparatively analyzed. Heterodimeric (αβ) bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi as well as monomeric luciferases of Vibrio harveyi and Luciola mingrelica (firefly) were used as substrates. In the presence of TF, thermally inactivated heterodimeric bacterial luciferases refold, while monomeric luciferases do not refold. These observations were made both in vivo (Escherichia coli ΔdnaKJ containing plasmids with tig gene) and in vitro (purified TF). Unlike TF, the DnaKJE chaperone system refolds both monomeric and heterodimeric luciferases with equal efficiency.     

5S rRNA-assisted DnaK refolding

Although accumulating evidence has revealed that most proteins can fold without the assistance of molecular chaperones, little attention has been paid to other types of chaperoning macromolecules. A variety of proteins interact with diverse RNA molecules in vivo, suggesting a potential role of RNAs for folding of their interacting proteins. Here we show that the in vitro refolding of a representative molecular chaperone, DnaK, an Escherichia coli homolog of Hsp70, could be assisted by its interacting 5S rRNA. The folding enhancement occurred in RNA concentration and its size dependent manner whereas neither the RNA with the reverse sequence of 5S rRNA nor the RNase pretreated 5S rRNA stimulated the folding in vitro. Based on our results, we propose that 5S rRNA could exert the chaperoning activity on DnaK during the folding process. The results suggest an interesting possibility that the folding of RNA-interacting proteins could be assisted by their cognate RNA ligands.     

Protein folding rates and thermodynamic stability are key determinants for interaction with the Hsp70 chaperone system

The Hsp70 family of molecular chaperones participates in vital cellular processes including the heat shock response and protein homeostasis. E. coli''s Hsp70, known as DnaK, works in concert with the DnaJ and GrpE co-chaperones (K/J/E chaperone system), and mediates cotranslational and post-translational protein folding in the cytoplasm. While the role of the K/J/E chaperones is well understood in the presence of large substrates unable to fold independently, it is not known if and how K/J/E modulates the folding of smaller proteins able to fold even in the absence of chaperones. Here, we combine experiments and computation to evaluate the significance of kinetic partitioning as a model to describe the interplay between protein folding and binding to the K/J/E chaperone system. First, we target three nonobligatory substrates, that is, proteins that do not require chaperones to fold. The experimentally observed chaperone association of these client proteins during folding is entirely consistent with predictions from kinetic partitioning. Next, we develop and validate a computational model (CHAMP70) that assumes kinetic partitioning of substrates between folding and interaction with K/J/E. CHAMP70 quantitatively predicts the experimentally measured interaction of RNase H^D as it refolds in the presence of various chaperones. CHAMP70 shows that substrates are posed to interact with K/J/E only if they are slow-folding proteins with a folding rate constant k_f <50 s⁻¹, and/or thermodynamically unstable proteins with a folding free energy ΔG⁰_UN ≥−2 kcal mol⁻¹. Hence, the K/J/E system is tuned to use specific protein folding rates and thermodynamic stabilities as substrate selection criteria.     

Low temperature of GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor DnaK

Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30 degrees C. Here we show that the synthetic lethality of deltatigdeltadnaK52 cells is abrogated either by growth below 30 degrees C or by overproduction of GroEL/GroES. At 23 degrees C deltatigdeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of deltatigdeltadnaK52 cells at 30 degrees C and suppressed protein aggregation including proteins >/= 60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates.     

Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor and DnaK

Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30 degrees C. Here we show that the synthetic lethality of DeltatigDeltadnaK52 cells is abrogated either by growth below 30 degrees C or by overproduction of GroEL/GroES. At 23 degrees C DeltatigDeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of DeltatigDeltadnaK52 cells at 30 degrees C and suppressed protein aggregation including proteins >/=60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates.     

Identification and Expression of the <Emphasis Type="Italic">tig</Emphasis> Gene Coding for Trigger Factor from Psychrophilic Bacteria with no Information of Genome Sequence Available

Trigger factor (TF) plays a key role as a molecular chaperone with a peptidyl-prolyl cis–trans isomerase (PPIase) activity by which cells promote folding of newly synthesized proteins coming out of ribosomes. Since psychrophilic bacteria grow at a quite low temperature, between 4 and 15°C, TF from such bacteria was investigated and compared with that of mesophilic bacteria E. coli in order to offer an explanation of cold-adaptation at a molecular level. Using a combination of gradient PCRs with homologous primers and LA PCR in vitro cloning technology, the tig gene was fully identified from Psychromonas arctica, whose genome sequence is not yet available. The resulting amino acid sequence of the TF was compared with other homologous TFs using sequence alignments to search for common domains. In addition, we have developed a protein expression system, by which TF proteins from P. arctica (PaTF) were produced by IPTG induction upon cloning the tig gene on expression vectors, such as pAED4. We have further examined the role of expressed psychrophilic PaTF on survival against cold treatment at 4°C. Finally, we have attempted the in vitro biochemical characterization of TF proteins with His-tags expressed in a pET system, such as the PPIase activity of PaTF protein. Our results demonstrate that the expressed PaTF proteins helped cells survive against cold environments in vivo and the purified PaTF in vitro display the functional PPIase activity in a concentration dependent manner.     

Functional adaptations of the bacterial chaperone trigger factor to extreme environmental temperatures

Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat‐induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.     

Molecular cloning, expression, and characterization of dnaK in Streptococcus pneumoniae

DnaK is known to be highly conserved in all species and is a major immunogen in Streptococcus pneumoniae. To elucidate the role of dnaK in S. pneumoniae, dnaK was cloned in Escherichia coli using a homologous dnaK probe generated by PCR. The His-tagged DnaK was overexpressed in soluble form and purified from E. coli. Alignment of the deduced DnaK amino acid sequence from nucleotide sequences of the cloned dnaK revealed high homology with DnaK analogs in E. coli (53%) and Staphylococcus aureus (73%). However, anti-pneumococcal DnaK antiserum did not crossreact with DnaK analogs in E. coli, S. aureus and human cells suggesting that pneumococcal DnaK might be a good candidate as a vaccine.