Hsp90, a team player in protein quality control and the stress response in bacteria.

SUMMARYHeat shock protein 90 (Hsp90) participates in proteostasis by facilitating protein folding, activation, disaggregation, prevention of aggregation, degradation, and protection against degradation of various cellular proteins. It is highly conserved from bacteria to humans. In bacteria, protein remodeling by Hsp90 involves collaboration with the Hsp70 molecular chaperone and Hsp70 cochaperones. In eukaryotes, protein folding by Hsp90 is more complex and involves collaboration with many Hsp90 cochaperones as well as Hsp70 and Hsp70 cochaperones. This review focuses primarily on bacterial Hsp90 and highlights similarities and differences between bacterial and eukaryotic Hsp90. Seminal research findings that elucidate the structure and the mechanisms of protein folding, disaggregation, and reactivation promoted by Hsp90 are discussed. Understanding the mechanisms of bacterial Hsp90 will provide fundamental insight into the more complex eukaryotic chaperone systems.

Corrigendum: Transcriptome reveals the role of the htpG gene in mediating antibiotic resistance through cell envelope modulation in Vibrio mimicus SCCF01.

[This corrects the article DOI: 10.3389/fmicb.2023.1295065.].

Bacterial heat shock protein genes during induction chemotherapy in pediatric patients with acute lymphoblastic leukemia.

Background: Heat shock proteins (HSP) protect cancer cells. Gastrointestinal bacteria contain HSP genes and can release extracellular vesicles which act as biological shuttles. Stress from treatment may result in a microbial community with more HSP genes, which could contribute to circulating HSP levels. Methods: The authors examined the abundance of five bacterial HSP genes pre-treatment and during induction in stool sequences from 30 pediatric acute lymphoblastic leukemia patients. Results: Decreased mean HTPG counts (p = 0.0024) pre-treatment versus induction were observed. During induction, HTPG, Shannon diversity and Bacteroidetes decreased (p = 7.5e-4; 1.1e-3; 8.6e-4), while DNAK and Firmicutes increased (p = 6.9e-3; 9.2e-4). Conclusion: Understanding microbial HSP gene community changes with treatment is the first step in determining if bacterial HSPs are important to the tumor microenvironment and leukemia treatment.

EspB and HtpG interact with the type III-A CRISPR/Cas system of Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis (MTB) has a type III-A clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system consisting of a Csm1-5 and CRISPR RNA (crRNA) complex involved in the defense against invading nucleic acids. However, CRISPR/Cas system in the MTB still is clearly unknown and needs to be further explored. Methods: In our work, two non-Cas system proteins EspB and HtpG protein were found and identified by LC-MS/MS. The effect of EspB and HtpG on Type III-A CRISPR/Cas System of M. tuberculosis was examined by using Plasmid interference assay and Co-immunoprecipitation analyses. We explored that EspB could interact with the crRNA RNP complex, but HtpG could inhibit the accumulation of the MTB Csm proteins and defense the mechanism of CRISPR/Cas system. Results: The proteins ESAT-6 secretion system-1(Esx-1) secreted protein B (EspB) and high-temperature protein G (HtpG), which were not previously associated with CRISPR/Cas systems, are involved in mycobacterial CRISPR/Cas systems with distinct functions. Conclusion: EspB is a novel crRNA-binding protein that interacts directly with the MTB crRNP complex. Meanwhile, HtpG influences the accumulation of MTB Csm proteins and EspB and interferes with the defense mechanism of the crRNP complex against foreign DNA in vivo. Thereby, our study not only leads to developing more precise clinical diagnostic tool to quickly detect for MTB infection, but also knows these proteins merits for TB biomarkers/vaccine candidates.

J-domain Proteins form Binary Complexes with Hsp90 and Ternary Complexes with Hsp90 and Hsp70.

Hsp90 and Hsp70 are highly conserved molecular chaperones that help maintain proteostasis by participating in protein folding, unfolding, remodeling and activation of proteins. Both chaperones are also important for cellular recovery following environmental stresses. Hsp90 and Hsp70 function collaboratively for the remodeling and activation of some client proteins. Previous studies using E. coli and S. cerevisiae showed that residues in the Hsp90 middle domain directly interact with a region in the Hsp70 nucleotide binding domain, in the same region known to bind J-domain proteins. Importantly, J-domain proteins facilitate and stabilize the interaction between Hsp90 and Hsp70 both in E. coli and S. cerevisiae. To further explore the role of J-domain proteins in protein reactivation, we tested the hypothesis that J-domain proteins participate in the collaboration between Hsp90 and Hsp70 by simultaneously interacting with Hsp90 and Hsp70. Using E. coli Hsp90, Hsp70 (DnaK), and a J-domain protein (CbpA), we detected a ternary complex containing all three proteins. The interaction involved the J-domain of CbpA, the DnaK binding region of E. coli Hsp90, and the J-domain protein binding region of DnaK where Hsp90 also binds. Additionally, results show that E. coli Hsp90 interacts with E. coli J-domain proteins, DnaJ and CbpA, and that yeast Hsp90, Hsp82, interacts with a yeast J-domain protein, Ydj1. Together these results suggest that the complexes may be transient intermediates in the pathway of collaborative protein remodeling by Hsp90 and Hsp70.

HtpG Is a Metal-Dependent Chaperone Which Assists the DnaK/DnaJ/GrpE Chaperone System of Mycobacterium tuberculosis via Direct Association with DnaJ2.

Protein folding is a crucial process in maintaining protein homeostasis, also known as proteostasis, in the cell. The requirement for the assistance of molecular chaperones in the appropriate folding of several proteins has already called into question the previously held view of spontaneous protein folding. These chaperones are highly ubiquitous cellular proteins, which not only help in mediating the proper folding of other nascent polypeptides but are also involved in refolding of the misfolded or the aggregated proteins. Hsp90 family proteins such as high-temperature protein G (HtpG) are abundant and ubiquitously expressed in both eukaryotic and prokaryotic cells. Although HtpG is known as an ATP-dependent chaperone protein in most organisms, function of this protein remains obscured in mycobacterial pathogens. Here, we aim to investigate significance of HtpG as a chaperone in the physiology of Mycobacterium tuberculosis. We report that M. tuberculosis HtpG (mHtpG) is a metal-dependent ATPase which exhibits chaperonin activity towards denatured proteins in coordination with the DnaK/DnaJ/GrpE chaperone system via direct association with DnaJ2. Increased expression of DnaJ1, DnaJ2, ClpX, and ClpC1 in a ΔhtpG mutant strain further suggests cooperativity of mHtpG with various chaperones and proteostasis machinery in M. tuberculosis. IMPORTANCE M. tuberculosis is exposed to variety of extracellular stressful conditions and has evolved mechanisms to endure and adapt to the adverse conditions for survival. mHtpG, despite being dispensable for M. tuberculosis growth under in vitro conditions, exhibits a strong and direct association with DnaJ2 cochaperone and assists the mycobacterial DnaK/DnaJ/GrpE (KJE) chaperone system. These findings suggest the potential role of mHtpG in stress management of the pathogen. Mycobacterial chaperones are responsible for folding of nascent protein as well as reactivation of protein aggregates. M. tuberculosis shows differential adaptive response subject to the availability of mHtpG. While its presence facilitates improved protein refolding via stimulation of the KJE chaperone activity, in the absence of mHtpG, M. tuberculosis enhances expression of DnaJ1/J2 cochaperones as well as Clp protease machinery for maintenance of proteostasis. Overall, this study provides a framework for future investigation to better decipher the mycobacterial proteostasis network in the light of stress adaptability and/or survival.

Mechanisms regulating the CRISPR-Cas systems.

The CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR associated proteins) is a prokaryotic system that enables sequence specific recognition and cleavage of nucleic acids. This is possible due to cooperation between CRISPR array which contains short fragments of DNA called spacers that are complimentary to the targeted nucleic acid and Cas proteins, which take part in processes of: acquisition of new spacers, processing them into their functional form as well as recognition and cleavage of targeted nucleic acids. The primary role of CRISPR-Cas systems is to provide their host with an adaptive and hereditary immunity against exogenous nucleic acids. This system is present in many variants in both Bacteria and Archea. Due to its modular structure, and programmability CRISPR-Cas system become attractive tool for modern molecular biology. Since their discovery and implementation, the CRISPR-Cas systems revolutionized areas of gene editing and regulation of gene expression. Although our knowledge on how CRISPR-Cas systems work has increased rapidly in recent years, there is still little information on how these systems are controlled and how they interact with other cellular mechanisms. Such regulation can be the result of both auto-regulatory mechanisms as well as exogenous proteins of phage origin. Better understanding of these interaction networks would be beneficial for optimization of current and development of new CRISPR-Cas-based tools. In this review we summarize current knowledge on the various molecular mechanisms that affect activity of CRISPR-Cas systems.

Transcriptome reveals the role of the htpG gene in mediating antibiotic resistance through cell envelope modulation in Vibrio mimicus SCCF01.

HtpG, a bacterial homolog of the eukaryotic 90 kDa heat-shock protein (Hsp90), represents the simplest member of the heat shock protein family. While the significance of Hsp90 in fungal and cancer drug resistance has been confirmed, the role of HtpG in bacterial antibiotic resistance remains largely unexplored. This research aims to investigate the impact of the htpG gene on antibiotic resistance in Vibrio mimicus. Through the creation of htpG gene deletion and complementation strains, we have uncovered the essential role of htpG in regulating the structural integrity of the bacterial cell envelope. Our transcriptomics analysis demonstrates that the deletion of htpG increases the sensitivity of V. mimicus to antimicrobial peptides, primarily due to upregulated lipopolysaccharide synthesis, reduced glycerophospholipid content, and weakened efflux pumps activity. Conversely, reduced sensitivity to ß-lactam antibiotics in the ΔhtpG strain results from decreased peptidoglycan synthesis and dysregulated peptidoglycan recycling and regulation. Further exploration of specific pathway components is essential for a comprehensive understanding of htpG-mediated resistance mechanisms, aiding in the development of antimicrobial agents. To our knowledge, this is the first effort to explore the relationship between htpG and drug resistance in bacteria.

Uncoupling the Hsp90 and DnaK chaperone activities revealed the in vivo relevance of their collaboration in bacteria.

Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major adenosine triphosphate (ATP)-dependent chaperone highly conserved from bacteria to eukaryotes. Recent studies have shown that bacterial Hsp90 is essential in some bacteria in stress conditions and that it participates in the virulence of pathogenic bacteria. In vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins; however, it is still unknown whether this collaboration is relevant in vivo with physiological substrates. Here, we used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the chaperone activities. We tested the mutants in vivo in two bacterial models in which Hsp90 has known physiological functions. We found that the Hsp90 point mutants were defective to support (1) growth under heat stress and activation of an essential Hsp90 client in the aquatic bacterium Shewanella oneidensis and (2) biosynthesis of the colibactin toxin involved in the virulence of pathogenic Escherichia coli. Our study therefore demonstrates the essentiality of the direct collaboration between Hsp90 and DnaK in vivo in bacteria to support client folding. It also suggests that this collaboration already functional in bacteria has served as an evolutionary basis for a more complex Hsp70-Hsp90 collaboration found in eukaryotes.

Involvement of the Heat Shock Protein HtpG of Salmonella Typhimurium in Infection and Proliferation in Hosts.

Salmonella Typhimurium is a common pathogen infecting the gastrointestinal tract of humans and animals, causing host gastroenteritis and typhoid fever. Heat shock protein (HtpG) as a molecular chaperone is involved in the various cellular processes of bacteria, especially under environmental stress. However, the potential association of HtpG with S. Typhimurium infection remains unknown. In this study, we clarified that HtpG could also play a role as an effector in S. Typhimurium infection. RNA-seq indicated that the flagellar assembly pathway, infection pathway, and chemotaxis pathway genes of S. Typhimurium were downregulated after the mutation of HtpG, which resulted in compromises of S. Typhimurium motility, biofilm formation, adhesion, invasion, and inflammation-inducing ability. In addition, HtpG recombinant protein was capable of promoting the proliferation of S. Typhimurium in host cells and the resultant inflammation. Collectively, our results illustrated an important role of HtpG in S. Typhimurium infection.

The Bacterial Hsp90 Chaperone: Cellular Functions and Mechanism of Action.

Heat shock protein 90 (Hsp90) is a molecular chaperone that folds and remodels proteins, thereby regulating the activity of numerous substrate proteins. Hsp90 is widely conserved across species and is essential in all eukaryotes and in some bacteria under stress conditions. To facilitate protein remodeling, bacterial Hsp90 collaborates with the Hsp70 molecular chaperone and its cochaperones. In contrast, the mechanism of protein remodeling performed by eukaryotic Hsp90 is more complex, involving more than 20 Hsp90 cochaperones in addition to Hsp70 and its cochaperones. In this review, we focus on recent progress toward understanding the basic mechanisms of bacterial Hsp90-mediated protein remodeling and the collaboration between Hsp90 and Hsp70. We describe the universally conserved structure and conformational dynamics of these chaperones and their interactions with one another and with client proteins. The physiological roles of Hsp90 in Escherichia coli and other bacteria are also discussed. We anticipate that the information gained from exploring the mechanism of the bacterial chaperone system will provide a framework for understanding the more complex eukaryotic Hsp90 system.

Bacterial RF3 senses chaperone function in co-translational folding.

Molecular chaperones assist with protein folding by interacting with nascent polypeptide chains (NCs) during translation. Whether the ribosome can sense chaperone defects and, in response, abort translation of misfolding NCs has not yet been explored. Here we used quantitative proteomics to investigate the ribosome-associated chaperone network in E. coli and the consequences of its dysfunction. Trigger factor and the DnaK (Hsp70) system are the major NC-binding chaperones. HtpG (Hsp90), GroEL, and ClpB contribute increasingly when DnaK is deficient. Surprisingly, misfolding because of defects in co-translational chaperone function or amino acid analog incorporation results in recruitment of the non-canonical release factor RF3. RF3 recognizes aberrant NCs and then moves to the peptidyltransferase site to cooperate with RF2 in mediating chain termination, facilitating clearance by degradation. This function of RF3 reduces the accumulation of misfolded proteins and is critical for proteostasis maintenance and cell survival under conditions of limited chaperone availability.

Bacterial Hsp90 Facilitates the Degradation of Aggregation-Prone Hsp70-Hsp40 Substrates.

In eukaryotes, the 90-kDa heat shock proteins (Hsp90s) are profusely studied chaperones that, together with 70-kDa heat shock proteins (Hsp70s), control protein homeostasis. In bacteria, however, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains poorly characterized. To uncover physiological processes that depend on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild-type (WT) Escherichia coli and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG), and ΔdnaKdnaJΔhtpG (ΔKJG). Whereas the ΔG mutant showed no detectable proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and expressed dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that the triple mutant ΔKJG showed higher levels of metabolic and respiratory enzymes than ΔKJ, suggesting that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates. Further in vivo experiments suggest that such Hsp90-mediated degradation possibly occurs through the HslUV protease.

Structural Communication between the E. coli Chaperones DnaK and Hsp90.

The 70 kDa and 90 kDa heat shock proteins Hsp70 and Hsp90 are two abundant and highly conserved ATP-dependent molecular chaperones that participate in the maintenance of cellular homeostasis. In Escherichia coli, Hsp90 (Hsp90Ec) and Hsp70 (DnaK) directly interact and collaborate in protein remodeling. Previous work has produced a model of the direct interaction of both chaperones. The locations of the residues involved have been confirmed and the model has been validated. In this study, we investigate the allosteric communication between Hsp90Ec and DnaK and how the chaperones couple their conformational cycles. Using elastic network models (ENM), normal mode analysis (NMA), and a structural perturbation method (SPM) of asymmetric and symmetric DnaK-Hsp90Ec, we extract biologically relevant vibrations and identify residues involved in allosteric signaling. When one DnaK is bound, the dominant normal modes favor biological motions that orient a substrate protein bound to DnaK within the substrate/client binding site of Hsp90Ec and release the substrate from the DnaK substrate binding domain. The presence of one DnaK molecule stabilizes the entire Hsp90Ec protomer to which it is bound. Conversely, the symmetric model of DnaK binding results in steric clashes of DnaK molecules and suggests that the Hsp90Ec and DnaK chaperone cycles operate independently. Together, this data supports an asymmetric binding of DnaK to Hsp90Ec.

The Pseudomonas aeruginosa HSP90-like protein HtpG regulates IL-8 expression through NF-κB/p38 MAPK and CYLD signaling triggered by TLR4 and CD91.

Pulmonary infection activates acute inflammatory responses by recruiting neutrophils to the infection site; this recruitment is promoted by interleukin-8 (IL-8). However, IL-8 production in response to Pseudomonas aeruginosa HtpG (PA1596), a homolog of heat shock protein 90, has yet not been characterized in detail. htpG expression in P. aeruginosa strain was elevated upon infection of host cells, and HtpG was released into bacterial culture supernatant. Treatment of dTHP-1 macrophages with recombinant HtpG (rHtpG) increased production of IL-8 in a dose- and time-dependent manner, and this effect was abolished by inhibition of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) p38 signaling. By contrast, the rHtpG-mediated production of IL-8 was increased by suppression of cylindromatosis (CYLD), suggesting that CYLD is a negative regulator of this pathway. The upregulation of expression was coordinated by signals transmitting through toll-like receptor 4 (TLR4) with the aid of CD91. Together, these observations suggest that P. aeruginosa HtpG activates NF-κB, CYLD, and p38 MAPK in a TLR4-and CD91-dependent manner, leading to stimulation of IL-8 production in macrophages.

Hsp90 of E. coli modulates assembly of FtsZ, the bacterial tubulin homolog.

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone involved in ATP-dependent client protein remodeling and activation. It also functions as a protein holdase, binding and stabilizing clients in an ATP-independent process. Hsp90 remodels over 300 client proteins and is essential for cell survival in eukaryotes. In bacteria, Hsp90 is a highly abundant protein, although very few clients have been identified and it is not essential for growth in many bacterial species. We previously demonstrated that in Escherichia coli, Hsp90 causes cell filamentation when expressed at high levels. Here, we have explored the cause of filamentation and identified a potentially important client of E. coli Hsp90 (Hsp90Ec), FtsZ. We observed that FtsZ, a bacterial tubulin homolog essential for cell division, fails to assemble into FtsZ rings (divisomes) in cells overexpressing Hsp90Ec Additionally, Hsp90Ec interacts with FtsZ and inhibits polymerization of FtsZ in vitro, in an ATP-independent holding reaction. The FtsZ-Hsp90Ec interaction involves residues in the client-binding region of Hsp90Ec and in the C-terminal tail of FtsZ, where many cell-division proteins and regulators interact. We observed that E. coli deleted for the Hsp90Ec gene htpG turn over FtsZ more rapidly than wild-type cells. Additionally, the length of ΔhtpG cells is reduced compared to wild-type cells. Altogether, these results suggest that Hsp90Ec is a modulator of cell division, and imply that the polypeptide-holding function of Hsp90 may be a biologically important chaperone activity.

Intermolecular Interactions between Hsp90 and Hsp70.

Members of the Hsp90 and Hsp70 families of molecular chaperones are imp\ortant for the maintenance of protein homeostasis and cellular recovery following environmental stresses, such as heat and oxidative stress. Moreover, the two chaperones can collaborate in protein remodeling and activation. In higher eukaryotes, Hsp90 and Hsp70 form a functionally active complex with Hop (Hsp90-Hsp70 organizing protein) acting as a bridge between the two chaperones. In bacteria, which do not contain a Hop homolog, Hsp90 and Hsp70, DnaK, directly interact during protein remodeling. Although yeast possesses a Hop-like protein, Sti1, Hsp90, and Hsp70 can directly interact in yeast in the absence of Sti1. Previous studies showed that residues in the middle domain of Escherichia coli Hsp90 are important for interaction with the J-protein binding region of DnaK. The results did not distinguish between the possibility that (i) these sites were involved in direct interaction and (ii) the residues in these sites participate in conformational changes which are transduced to other sites on Hsp90 and DnaK that are involved in the direct interaction. Here we show by crosslinking experiments that the direct interaction is between a site in the middle domain of Hsp90 and the J-protein binding site of Hsp70 in both E. coli and yeast. Moreover, J-protein promotes the Hsp70-Hsp90 interaction in the presence of ATP, likely by converting Hsp70 into the ADP-bound conformation. The identification of the protein-protein interaction site is anticipated to lead to a better understanding of the collaboration between the two chaperones in protein remodeling.

Cas3-stimulated runaway replication of modified ColE1 plasmids in Escherichia coli is temperature dependent.

The clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system constitutes an adaptive immunity system of prokaryotes against mobile genetic elements using a CRISPR RNA (crRNA)-mediated interference mechanism. In Type I CRISPR-Cas systems, crRNA guided by a Cascade complex recognises the matching target DNA and promotes an R-loop formation, RNA-DNA hybrid. The helicase-nuclease Cas3 protein is then recruited to the Cascade/R-loop complex where it nicks and degrades DNA. The Cas3 activity in CRISPR-Cas immunity is reduced in Δhns cells at 37°C for unknown reasons. Cas3 can also influence regulation of plasmid replication and promote uncontrolled ('runaway') replication of ColE1 plasmids independently of other CRISPR-Cas components, requiring only its helicase activity. In this work we wanted to test whether Cas3-stimulated uncontrolled plasmid replication is affected by the temperature in Δhns and/or ΔhtpG mutants. We found that Cas3-stimulated uncontrolled plasmid replication occurs only at 37°C, irrespective of the genotype of the analysed mutants, and dependent on Cas3 helicase function. We also found that plasmid replication was strongly reduced by the hns mutation at 30°C and that Cas3 could interfere with T4 phage replication at both incubation temperatures.

Dual RNA-Seq Unveils Pseudomonas plecoglossicida htpG Gene Functions During Host-Pathogen Interactions With Epinephelus coioides.

Pseudomonas plecoglossicida is a temperature-dependent opportunistic pathogen which is associated with a variety of diseases in fish. During the development of "white nodules" disease, the expression of htpG in P. plecoglossicida was found to be significantly up-regulated at its virulent temperature of 18°C. The infection of htpG-RNAi strain resulted in the onset time delay, reduction in mortality and infection symptoms in spleen of Epinephelus coioides, and affected the bacterial tissue colonization. In order to reveal the effect of htpG silencing of P. plecoglossicida on the virulence regulation in P. plecoglossicida and immune response in E. coioides, dual RNA-seq was performed and a pathogen-host integration network was constructed. Our results showed that infection induced the expression of host genes related to immune response, but attenuated the expression of bacterial virulence genes. Novel integration was found between host immune genes and bacterial virulence genes, while IL6, IL1R2, IL1B, and TLR5 played key roles in the network. Further analysis with GeneMANIA indicated that flgD and rplF might play key roles during the htpG-dependent virulence regulation, which was in accordance with the reduced biofilm production, motility and virulence in htpG-RNAi strain. Meanwhile, IL6 and IL1B were found to play key roles during the defense against P. plecoglossicida, while CELA2, TRY, CPA1, CPA2, and CPB1 were important targets for P. plecoglossicida attacking to the host.

Hsp90 and Hsp70 chaperones: Collaborators in protein remodeling.

Heat shock proteins 90 (Hsp90) and 70 (Hsp70) are two families of highly conserved ATP-dependent molecular chaperones that fold and remodel proteins. Both are important components of the cellular machinery involved in protein homeostasis and participate in nearly every cellular process. Although Hsp90 and Hsp70 each carry out some chaperone activities independently, they collaborate in other cellular remodeling reactions. In eukaryotes, both Hsp90 and Hsp70 function with numerous Hsp90 and Hsp70 co-chaperones. In contrast, bacterial Hsp90 and Hsp70 are less complex; Hsp90 acts independently of co-chaperones, and Hsp70 uses two co-chaperones. In this review, we focus on recent progress toward understanding the basic mechanisms of Hsp90-mediated protein remodeling and the collaboration between Hsp90 and Hsp70, with an emphasis on bacterial chaperones. We describe the structure and conformational dynamics of these chaperones and their interactions with each other and with client proteins. The physiological roles of Hsp90 in Escherichia coli and other bacteria are also discussed. We anticipate that the information gained from exploring the mechanism of the bacterial chaperone system will provide the groundwork for understanding the more complex eukaryotic Hsp90 system and its modulation by Hsp90 co-chaperones.