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.

Directed evolution of SecB chaperones toward toxin-antitoxin systems.

SecB chaperones assist protein export in bacteria. However, certain SecB family members have diverged to become specialized toward the control of toxin-antitoxin (TA) systems known to promote bacterial adaptation to stress and persistence. In such tripartite TA-chaperone (TAC) systems, the chaperone was shown to assist folding and to prevent degradation of its cognate antitoxin, thus facilitating inhibition of the toxin. Here, we used both the export chaperone SecB of Escherichia coli and the tripartite TAC system of Mycobacterium tuberculosis as a model to investigate how generic chaperones can specialize toward the control of TA systems. Through directed evolution of SecB, we have identified and characterized mutations that specifically improve the ability of SecB to control our model TA system without affecting its function in protein export. Such a remarkable plasticity of SecB chaperone function suggests that its substrate binding surface can be readily remodeled to accommodate specific clients.

Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane.

Nascent polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytosolic, integral membrane or exported proteins. Extensive genetic and biochemical analyses have significantly expanded our knowledge of chaperone tasking throughout this process. In bacteria, it is known that the folding of newly-synthesized cytosolic proteins is mainly orchestrated by three highly conserved molecular chaperones, namely Trigger Factor (TF), DnaK (HSP70) and GroEL (HSP60). Yet, it has been reported that these major chaperones are strongly involved in protein translocation pathways as well. This review describes such essential molecular chaperone functions, with emphasis on both the biogenesis of inner membrane proteins and the post-translational targeting of presecretory proteins to the Sec and the twin-arginine translocation (Tat) pathways. Critical interplay between TF, DnaK, GroEL and other molecular chaperones and targeting factors, including SecB, SecA, the signal recognition particle (SRP) and the redox enzyme maturation proteins (REMPs) is also discussed. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

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.

Acid stress response in Escherichia coli: mechanism of regulation of gadA transcription by RcsB and GadE.

Escherichia coli can survive extreme acid stress for several hours. The most efficient acid resistance system is based on glutamate decarboxylation by the GadA and GadB decarboxylases and the import of glutamate via the GadC membrane protein. The expression of the corresponding genes is controlled by GadE, the central activator of glutamate-dependent acid resistance (GDAR). We have previously shown by genetic approaches that as well as GadE, the response regulator of the Rcs system, RcsB is absolutely required for control of gadA/BC transcription. In the presence of GadE, basal activity of RcsB stimulates the expression of gadA/BC, whereas activation of RcsB leads to general repression of the gad genes. We report here the results of various in vitro assays that show RcsB to regulate by direct binding to the gadA promoter region. Furthermore, activation of gadA transcription requires a GAD box and binding of an RcsB/GadE heterodimer. In addition, we have identified an RcsB box, which lies just upstream of the -10 element of gadA promoter and is involved in repression of this operon.

Copper Induces Protein Aggregation, a Toxic Process Compensated by Molecular Chaperones.

Copper is well known for its antimicrobial and antiviral properties. Under aerobic conditions, copper toxicity relies in part on the production of reactive oxygen species (ROS), especially in the periplasmic compartment. However, copper is significantly more toxic under anaerobic conditions, in which ROS cannot be produced. This toxicity has been proposed to arise from the inactivation of proteins through mismetallations. Here, using the bacterium Escherichia coli, we discovered that copper treatment under anaerobic conditions leads to a significant increase in protein aggregation. In vitro experiments using E. coli lysates and tightly controlled redox conditions confirmed that treatment with Cu+ under anaerobic conditions leads to severe ROS-independent protein aggregation. Proteomic analysis of aggregated proteins revealed an enrichment of cysteine- and histidine-containing proteins in the Cu+-treated samples, suggesting that nonspecific interactions of Cu+ with these residues are likely responsible for the observed protein aggregation. In addition, E. coli strains lacking the cytosolic chaperone DnaK or trigger factor are highly sensitive to copper stress. These results reveal that bacteria rely on these chaperone systems to protect themselves against Cu-mediated protein aggregation and further support our finding that Cu toxicity is related to Cu-induced protein aggregation. Overall, our work provides new insights into the mechanism of Cu toxicity and the defense mechanisms that bacteria employ to survive. IMPORTANCE With the increase of antibiotic drug resistance, alternative antibacterial treatment strategies are needed. Copper is a well-known antimicrobial and antiviral agent; however, the underlying molecular mechanisms by which copper causes cell death are not yet fully understood. Herein, we report the finding that Cu+, the physiologically relevant copper species in bacteria, causes widespread protein aggregation. We demonstrate that the molecular chaperones DnaK and trigger factor protect bacteria against Cu-induced cell death, highlighting, for the first time, the central role of these chaperones under Cu+ stress. Our studies reveal Cu-induced protein aggregation to be a central mechanism of Cu toxicity, a finding that will serve to guide future mechanistic studies and drug development.

Insights into the extracytoplasmic stress response of Xanthomonas campestris pv. campestris: role and regulation of {sigma}E-dependent activity.

Xanthomonas campestris pv. campestris is an epiphytic bacterium that can become a vascular pathogen responsible for black rot disease of crucifers. To adapt gene expression in response to ever-changing habitats, phytopathogenic bacteria have evolved signal transduction regulatory pathways, such as extracytoplasmic function (ECF) σ factors. The alternative sigma factor σ(E), encoded by rpoE, is crucial for envelope stress response and plays a role in the pathogenicity of many bacterial species. Here, we combine different approaches to investigate the role and mechanism of σ(E)-dependent activation in X. campestris pv. campestris. We show that the rpoE gene is organized as a single transcription unit with the anti-σ gene rseA and the protease gene mucD and that rpoE transcription is autoregulated. rseA and mucD transcription is also controlled by a highly conserved σ(E)-dependent promoter within the σ(E) gene sequence. The σ(E)-mediated stress response is required for stationary-phase survival, resistance to cadmium, and adaptation to membrane-perturbing stresses (elevated temperature and ethanol). Using microarray technology, we started to define the σ(E) regulon of X. campestris pv. campestris. These genes encode proteins belonging to different classes, including periplasmic or membrane proteins, biosynthetic enzymes, classical heat shock proteins, and the heat stress σ factor σ(H). The consensus sequence for the predicted σ(E)-regulated promoter elements is GGAACTN(15-17)GTCNNA. Determination of the rpoH transcription start site revealed that rpoH was directly regulated by σ(E) under both normal and heat stress conditions. Finally, σ(E) activity is regulated by the putative regulated intramembrane proteolysis (RIP) proteases RseP and DegS, as previously described in many other bacteria. However, our data suggest that RseP and DegS are not only dedicated to RseA cleavage and that the proteolytic cascade of RseA could involve other proteases.

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.

J-Domain Proteins in Bacteria and Their Viruses.

Molecular chaperones maintain cellular protein homeostasis by acting at almost every step in protein biogenesis pathways. The DnaK/HSP70 chaperone has been associated with almost every known essential chaperone functions in bacteria. To act as a bona fide chaperone, DnaK strictly relies on essential co-chaperone partners known as the J-domain proteins (JDPs, DnaJ, Hsp40), which preselect substrate proteins for DnaK, confer its specific cellular localization, and stimulate both its weak ATPase activity and substrate transfer. Remarkably, genome sequencing has revealed the presence of multiple JDP/DnaK chaperone/co-chaperone pairs in a number of bacterial genomes, suggesting that certain pairs have evolved toward more specific functions. In this review, we have used representative sets of bacterial and phage genomes to explore the distribution of JDP/DnaK pairs. Such analysis has revealed an unexpected reservoir of novel bacterial JDPs co-chaperones with very diverse and unexplored function that will be discussed.

Emerging fields in chaperone proteins: A French workshop.

The "Bioénergétique et Ingénierie des Protéines (BIP)" laboratory, CNRS (France), organized its first French workshop on molecular chaperone proteins and protein folding in November 2017. The goal of this workshop was to gather scientists working in France on chaperone proteins and protein folding. This initiative was a great success with excellent talks and fruitful discussions. The highlights were on the description of unexpected functions and post-translational regulation of known molecular chaperones (such as Hsp90, Hsp33, SecB, GroEL) and on state-of-the-art methods to tackle questions related to this theme, including Cryo-electron microscopy, Nuclear Magnetic Resonance (NMR), Electron Paramagnetic Resonance (EPR), simulation and modeling. We expect to organize a second workshop in two years that will include more scientists working in France in the chaperone field.

Multilevel interaction of the DnaK/DnaJ(HSP70/HSP40) stress-responsive chaperone machine with the central metabolism.

Networks of molecular chaperones maintain cellular protein homeostasis by acting at nearly every step in the biogenesis of proteins and protein complexes. Herein, we demonstrate that the major chaperone DnaK/HSP70 of the model bacterium Escherichia coli is critical for the proper functioning of the central metabolism and for the cellular response to carbon nutrition changes, either directly or indirectly via the control of the heat-shock response. We identified carbon sources whose utilization was positively or negatively affected by DnaK and isolated several central metabolism genes (among other genes identified in this work) that compensate for the lack of DnaK and/or DnaK/Trigger Factor chaperone functions in vivo. Using carbon sources with specific entry points coupled to NMR analyses of real-time carbon assimilation, metabolic coproducts production and flux rearrangements, we demonstrate that DnaK significantly impacts the hierarchical order of carbon sources utilization, the excretion of main coproducts and the distribution of metabolic fluxes, thus revealing a multilevel interaction of DnaK with the central metabolism.

The glutamate-dependent acid resistance system in Escherichia coli: essential and dual role of the His-Asp phosphorelay RcsCDB/AF.

The RcsCDB signal transduction system is an atypical His-Asp phosphorelay. Notably, the response regulator RcsB can be activated either by phosphorylation through the RcsCD pathway or by an accessory cofactor RcsA. Although conserved in Enterobacteriaceae, the role of this system in adaptation to environmental stress conditions is largely unknown. This study reveals that the response regulator RcsB is essential to glutamate-dependent acid resistance, a condition pertinent to the lifestyle of Escherichia coli. The requirement for RcsB is independent of its activation by either the RcsCD or the RcsA pathway. The basal activity of RcsB appears to be necessary and sufficient for acid resistance. The sensitivity of the rcsB strain to low pH is correlated to a strong reduction of the expression of the glutamate decarboxylase genes, gadA and gadB, during the stationary phase of growth. This effect on gadA/B expression is not mediated by the general stress sigma factor RpoS, but does require a functional gadE allele and the previously identified GadE box. Therefore activation of gadAB expression and acid resistance absolutely requires both GadE and RcsB. In contrast, an increase in RcsB activity through the activation of the RcsCD phosphorelay or the RcsA pathway or through overproduction of the protein leads to general repression of the expression of the gad genes and a corresponding reduction in acid resistance.

RcsF is an outer membrane lipoprotein involved in the RcsCDB phosphorelay signaling pathway in Escherichia coli.

The RcsCDB signal transduction system is an atypical His-Asp phosphorelay conserved in gamma-proteobacteria. Besides the three proteins directly involved in the phosphorelay, two proteins modulate the activity of the system. One is RcsA, which can stimulate the activity of the response regulator RcsB independently of the phosphorelay to regulate a subset of RcsB targets. The other is RcsF, a putative outer membrane lipoprotein mediating the signaling to the sensor RcsC. How RcsF transduces the signal to RcsC is unknown. Although the molecular and physiological signals remain to be identified, the common feature among the reported Rcs-activating conditions is perturbation of the envelope. As an initial step to explore the RcsF-RcsC functional relationship, we demonstrate that RcsF is an outer membrane lipoprotein oriented towards the periplasm. We also report that a null mutation in surA, a gene required for correct folding of periplasmic proteins, activates the Rcs pathway through RcsF. In contrast, activation of this pathway by overproduction of the membrane chaperone-like protein DjlA does not require RcsF. Conversely, activation of the pathway by RcsF overproduction does not require DjlA either, indicating the existence of two independent signaling pathways toward RcsC.

Osmotic regulation of the Escherichia coli bdm (biofilm-dependent modulation) gene by the RcsCDB His-Asp phosphorelay.

The RcsCDB His-Asp phosphorelay is shown to positively regulate the bdm (biofilm-dependent modulation) and sra (stationary-phase-induced ribosome-associated protein) genes in Escherichia coli. The regulation is direct and requires an RcsB box next to the bdm -35 element. In addition, bdm is shown to be activated by osmotic shock in an Rcs-dependent way.

Multistress regulation in Escherichia coli: expression of osmB involves two independent promoters responding either to sigmaS or to the RcsCDB His-Asp phosphorelay.

Transcription of the Escherichia coli osmB gene is induced by several stress conditions. osmB is expressed from two promoters, osmBp1 and osmBp2. The downstream promoter, osmBp2, is induced after osmotic shock or upon entry into stationary phase in a sigma(S)-dependent manner. The upstream promoter, osmBp1, is independent of sigma(S) and is activated by RcsB, the response regulator of the His-Asp phosphorelay signal transduction system RcsCDB. RcsB is responsible for the induction of osmBp1 following treatment with chlorpromazine. Activation of osmBp1 by RcsB requires a sequence upstream of its -35 element similar to the RcsB binding site consensus, suggesting a direct regulatory role. osmB appears as another example of a multistress-responsive gene whose transcription involves both a sigma(S)-dependent promoter and a second one independent of sigma(S) but controlled by stress-specific transcription factors.

Escherichia coli acid resistance: cAMP receptor protein and a 20 bp cis-acting sequence control pH and stationary phase expression of the gadA and gadBC glutamate decarboxylase genes.

Acid resistance is an important feature of both pathogenic and non-pathogenic Escherichia coli. It enables survival in the acidic regions of mammalian gastrointestinal tracts and is largely responsible for the small number of bacteria required for infection/colonization. Three systems of acid resistance have been identified, the most efficient of which requires glutamic acid during pH 2 acid challenge. Three proteins associated with glutamate-dependent acid resistance have been identified. They are glutamate decarboxylase (encompassing two isozymes encoded by gadA and gadB) and a putative glutamate:gamma-amino butyric acid antiporter (encoded by gadC). The results confirm that the GadA and GadB proteins increase in response to stationary phase and low environmental pH. The levels of these proteins correspond to concomitant changes in gadA and gadBC mRNA levels. Fusions between lacZ and the gadA and gadBC operons indicate that this control occurs at the transcriptional level. Western blot, Northern blot and fusion analyses reveal that regulation of these genes is complex. Expression in rich media is restricted to stationary phase. However, in minimal media, acid pH alone can trigger induction in exponential or stationary phase cells. Despite this differential control, there is only one transcriptional start site for each gene. Expression in rich media is largely dependent on the alternate sigma factor sigma(S) and is repressed by the cAMP receptor protein (CRP). In contrast, sigma(S) has only a minor role in gad transcription in cells grown in minimal media. Deletions of the regulatory region upstream of gadA provided evidence that a 20 bp conserved region located 50 bp from the transcriptional start of both operons is required for expression.

Acid resistance systems required for survival of Escherichia coli O157:H7 in the bovine gastrointestinal tract and in apple cider are different.

Escherichia coli O157:H7 is a highly acid-resistant food-borne pathogen that survives in the bovine and human gastrointestinal tracts and in acidic foods such as apple cider. This property is thought to contribute to the low infectious dose of the organism. Three acid resistance (AR) systems are expressed in stationary-phase cells. AR system 1 is sigma(S) dependent, while AR systems 2 and 3 are glutamate and arginine dependent, respectively. In this study, we sought to determine which AR systems are important for survival in acidic foods and which are required for survival in the bovine intestinal tract. Wild-type and mutant E. coli O157:H7 strains deficient in AR system 1, 2, or 3 were challenged with apple cider and inoculated into calves. Wild-type cells, adapted at pH 5.5 in the absence of glucose (AR system 1 induced), survived well in apple cider. Conversely, the mutant deficient in AR system 1, shown previously to survive poorly in calves, was susceptible to apple cider (pH 3.5), and this sensitivity was shown to be caused by low pH. Interestingly, the AR system 2-deficient mutant survived in apple cider at high levels, but its shedding from calves was significantly decreased compared to that of wild-type cells. AR system 3-deficient cells survived well in both apple cider and calves. Taken together, these results indicate that E. coli O157:H7 utilizes different acid resistance systems based on the type of acidic environment encountered.

RcsCDB His-Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli.

The genes involved in flagellum synthesis, motility and chemotaxis in Escherichia coli are expressed in a hierarchical fashion. At the top of the hierarchy lies the master regulator FlhDC, required for the expression of the whole set of genes. The operon flhDC is controlled by numerous regulators including H-NS, CRP, EnvZ/OmpR, QseBC and LrhA. In the present work, we report that the flhDC operon is also negatively regulated by the His-Asp phosphorelay system RcsCDB. The regulation is potentiated by the RcsB cofactor RcsA. Genetic analysis indicates that an RcsAB box, located downstream of the promoter, is required for the regulation. The binding of RcsB and RcsA to this site was demonstrated by gel retardation and DNase I protection assays. In addition, mutation analysis suggests that RcsA-specific determinants lie in the right part of the 'RcsAB box'.