Genes grpE Research Articles

An Escherichia coli gene (yaeO) suppresses temperature-sensitive mutations in essential genes by modulating Rho-dependent transcription termination.

An extragenic multicopy suppressor of the cell division inhibition caused by a MalE-MinE fusion protein in Escherichia coli has been mapped and identified as yaeO, one of the two short open reading frames (ORFs) of an operon located at 4.6 min. Overexpressed yaeO also suppressed some temperature-sensitive mutations in division genes ftsA and ftsQ, in chaperone gene groEL and in co-chaperone gene grpE. Gene yaeO, whose expression is regulated by growth rate, codes for a 9 kDa acidic protein with no obvious resemblance to other proteins. Transcription termination protein Rho co-purified with a histidine-tagged derivative of YaeO protein on Ni2+-NTA agarose columns in a manner that suggested direct YaeO-Rho interaction. In vivo, yaeO expression reduced termination at rho-dependent bacteriophage terminator tL1 and at the terminator of autogenously regulated gene rho. The suppression of temperature-sensitive phenotypes was a consequence of anti-termination, as it could be mimicked by a Prho::Tn10 mutation that reduces the expression and activity of gene rho. Our data indicate that the suppression is not caused by overexpression of the mutated genes, but presumably by indirect stabilization of the mutated proteins.

Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli.

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.

Cloning, sequencing, and expression of dnaK-operon proteins from the thermophilic bacterium Thermus thermophilus

We propose that the dnaK operon of Thermus thermophilus HB8 is composed of three functionally linked genes: dnaK, grpE, and dnaJ. The dnaK and dnaJ gene products are most closely related to their cyanobacterial homologs. The DnaK protein sequence places T. thermophilus in the plastid Hsp70 subfamily. In contrast, the grpE translated sequence is most similar to GrpE from Clostridium acetobutylicum, a Gram-positive anaerobic bacterium. A single promoter region, with homology to the Escherichia coli consensus promoter sequences recognized by the σ 70 and σ 32 transcription factors, precedes the postulated operon. This promoter is heat-shock inducible. The dnaK mRNA level increased more than 30 times upon 10 min of heat shock (from 70°C to 85°C). A strong transcription terminating sequence was found between the dnaK and grpE genes. The individual genes were cloned into pET expression vectors and the thermophilic proteins were overproduced at high levels in E. coli and purified to homogeneity. The recombinant T. thermophilus DnaK protein was shown to have a weak ATP-hydrolytic activity, with an optimum at 90°C. The ATPase was stimulated by the presence of GrpE and DnaJ. Another open reading frame, coding for ClpB heat-shock protein, was found downstream of the dnaK operon.

Cloning and sequencing of the dnaK and grpE genes of Legionella pneumophila

A 4.4-kb DNA fragment from Legionella pneumophila ( Lp) was isolated, which could complement an Escherichia coli ( Ec) dnaK ts mutant, HC4102. Nucleotide sequence analysis of the region revealed two complete open reading frames (ORFs) encoding both a predicted DnaK protein of 644 aa and a predicted GrpE protein of 199 aa, and also the 5′-end of the predicted dnaJ gene organized in the order of grpE– dnaK– dnaJ. Consensus heat shock (HS) promoter sequences were identified upstream of the start of both grpE and dnaK transcripts. However, no obvious promoter sequences were detected upstream of dnaJ. The transcription start points of grpE and dnaK were determined by primer extension analysis and the amount of each of the transcripts increased four- to eightfold after HS.

K + is an indispensable cofactor for GrpE stimulation of ATPase activity of DnaK·DnaJ complex from Thermus thermophilus

K + is an indispensable cofactor for ATPase activity of eukaryotic cytosolic Hsp70 chaperone systems which lack a GrpE homolog. In the case of the bacterial Hsp70 (DnaK) system, GrpE, a nucleotide exchange factor, stimulates ATPase activity but little is known about the effect of K +. Here, we have cloned a grpE gene from a thermophile, Thermus thermophilus, and purified a homodimeric GrpE protein. Using proteins of this bacterium, we found that the GrpE stimulation of ATPase activity of DnaK·DnaJ complex was absolutely dependent on the presence of K +.

Increased transcripts of the dnaK locus genes in Methanosarcina mazei S-6 exposed to supraoptimal concentrations of ammonia

The effect of supraoptimal concentrations of ammonia on cell growth and stress genes of the dnaK locus was investigated in Methanosarcina mazei S-6, a methanogen important for anaerobic digestion in bioreactors. When the concentration of NH 4Cl was increased 25 times above the optimal for growth, the lag phase lengthened, growth occurred at a decreased rate, and a stress response was induced – the transcripts from the stress genes grpE, dnaK, and dnaJ increased, whereas that of the adjacent genes orf11-trkA decreased. In contrast, a 10-fold elevation of the NH 4Cl concentration, which had only a slight stimulating effect on the stress genes, augmented the orf11-trkA transcript. These findings show that ammonia at certain levels is a cell stressor for M. mazei S-6.

Transcriptional analysis of the Streptococcus mutans hrcA, grpE and dnaK genes and regulation of expression in response to heat shock and environmental acidification

The dental pathogen Streptococcus mutans persists and causes diseases in highly dynamic environments and gains a selective ecological advantage in environmental conditions that frequently exceed the limits for growth of the organism, particularly with regard to environmental pH. The goal of this study was to begin a molecular genetic analysis of a major stress protein, DnaK/Hsp70, to begin to understand how stress responses are regulated in this lactic acid bacterium and to establish a relationship between dnaK gene expression and exposure to acidic environments. Cloning and nucleotide sequence analysis revealed that the dnaK gene is preceded by, and is in an operon-like arrangement with, the hrcA and grpE genes, although intergenic spacing was unlike that described in other bacteria. An inverted repeat (a CIRCE element) was identified by sequence analysis and, using primer extensions, a heat shock-responsive, sigmaA-type promoter, P1, 5' to the hrcA gene, and a sigmaB-type promoter, 5' to the grpE translational start site, were identified. No promoters were detected between grpE and dnaK. A strain carrying a strongly polar insertion in the hrcA gene had markedly diminished levels of dnaK mRNA, indicating that dnaK was transcribed as part of an operon from P1, and to a lesser extent from P2. Results from physiological manipulation of S. mutans in continuous chemostat culture demonstrated that steadystate levels of S. mutans dnaK mRNA and DnaK protein were (i) increased in response to acid shock; (ii) elevated in acid 'adapted' cells; and (iii) induced in response to alkali shock of acid 'adapted' cells. In all cases, increased amounts of dnaK mRNA could be correlated with enhanced transcription from P1. This study provides the first detailed analysis of the expression of a heat shock gene from an oral isolate, and the evidence provided suggests that sigmaB-like promoters may also be involved in class I heat shock gene expression in some Gram-positive organisms.

Isolation and characterization of a dnaK genomic locus in a halotolerant cyanobacterium Aphanothece halophytica.

We cloned and characterized a genomic locus encoding a distinct member of the DnaK/Hsp70 family of molecular chaperones, dnaK1, from the halotolerant cyanobacterium Aphanothece halophytica. Co-expression of dnaK1 with a plant plastocyanin precursor in Escherichia coli resulted in a dramatic increase in the solubility of the plant protein. This indicates that A. halophytica dnaK1 encodes a functional protein possessing functions assigned to DnaK/Hsp70 chaperone members. The A. halophytica dnaK1 locus also encompasses grpE and dnaJ homologue genes in the order grpE-dnaK1-dnaJ. The transcript content of dnaK1 increased strongly upon subjecting cyanobacterial cells to heat stress. Northern analyses using specific probes indicated transcript species of 2.8, 2.2, 1.3, and 0.7 kb, which comprised grpE-dnaK1, dnaK1, dnaJ, and grpE, respectively. This indicates the presence of different terminators and/or heat stress promoters in this locus. Both dnaK1 transcript and protein levels increased in cyanobacterial cells transferred to hyperosmotic environments, suggesting a role of DnaK1 in the protection and/or recovery of A. halophytica from this particular stress.

Structure-function analysis of the Escherichia coli GrpE heat shock protein.

We have isolated various missense mutations in the essential grpE gene of Escherichia coli based on the inability to propagate bacteriophage lambda. To better understand the biochemical mechanisms of GrpE action in various biological processes, six mutant proteins were overexpressed and purified. All of them, GrpE103, GrpE66, GrpE2/280, GrpE17, GrpE13a and GrpE25, have single amino acid substitutions located in highly conserved regions throughout the GrpE sequence. The biochemical defects of each mutant GrpE protein were identified by examining their abilities to: (i) support in vitro lambda DNA replication; (ii) stimulate the weak ATPase activity of DnaK; (iii) dimerize and oligomerize, as judged by glutaraldehyde crosslinking and HPLC size chromatography; (iv) interact with wild-type DnaK protein using either an ELISA assay, glutaraldehyde crosslinking or HPLC size chromatography. Our results suggest that GrpE can exist in a dimeric or oligomeric form, depending on its relative concentration, and that it dimerizes/oligomerizes through its N-terminal region, most likely through a computer predicted coiled-coil region. Analysis of several mutant GrpE proteins indicates that an oligomer of GrpE is the most active form that interacts stably with DnaK and that the interaction is vital for GrpE biological function. Our results also demonstrate that both the N-terminal and C-terminal regions are important for GrpE function in lambda DNA replication and its co-chaperone activity with DnaK.

Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE

In response to elevated temperature, both prokaryotic and eukaryotic cells increase expression of a small family of chaperones. The regulatory network that functions to control the transcription of the heat shock genes in bacteria includes unique structural motifs in the promoter region of these genes and the expression of alternate sigma factors. One of the conserved structural motifs, the inverted repeat CIRCE element, is found in the 5' region of many heat shock operons, including the Caulobacter crescentus groESL operon. We report the identification of another C. crescentus heat shock operon containing two genes, hrcA (hrc for heat shock regulation at CIRCE elements) and a grpE homolog. Disruption of the hrcA gene, homologs of which are also found upstream of grpE in other bacteria, increased transcription of the groESL operon, and this effect was dependent on the presence of an intact CIRCE element. This suggests a role for HrcA in negative regulation of heat shock gene expression. We identified a major promoter transcribing both hrcA and grpE and a minor promoter located within the hrcA coding sequence just upstream of grpE. Both promoters were heat shock inducible, with maximal expression 10 to 20 min after heat shock. Both promoters were also expressed constitutively throughout the cell cycle under physiological conditions. C. crescentus GrpE, shown to be essential for viability at low and high temperatures, complemented an Escherichia coli delta grpE strain in spite of significant differences in the N- and C-terminal regions of these two proteins, demonstrating functional conservation of this important stress protein.

Isolation and characterization of point mutations in the Escherichia coli grpE heat shock gene

The Escherichia coli grpE gene (along with dnaK, dnaJ, groEL, and groES) was originally identified as one of the host factors required for phage lambda growth. The classical grpE280 mutation was the only grpE mutation that resulted from the initial screen and shown to specifically block the initiation of lambda DNA replication. Here we report the isolation of several new grpE missense mutations, again using phage lambda resistance as a selection. All mutants fall into two groups based on their temperature-dependent phenotype for lambda growth. Members of the first group (I), including grpE17 and grpE280, which was obtained again, are resistant to lambda growth at both 30 and 42 degrees C. Members of the second group (II), including grpE25, grpE66, grpE103, grpE13a, grpE57b, and grpE61, are sensitive to lambda growth at 30 degrees C but resistant at 42 degrees C. All mutations are recessive, since an E. coli grpE null mutant strain carrying these mutant alleles on low-copy-number plasmids are sensitive to infection by the lambda grpE+ transducing phage. Both group I and group II mutants are temperature sensitive for E. coli growth above 42 degrees C. The nucleotide changes were identified by sequencing analyses and shown to be dispersed throughout the latter 75% of the grpE coding region. Most of the amino acid changes occur at conserved residues, as judged by sequence comparisons between E. coli and other bacterial and yeast GrpE homologs. The isolation of these new mutations is the first step toward a structure-function analysis of the GrpE protein.

Identification of a grpE Heat-shock Gene Homolog in the Archeon Methanosarcina mazie

A grpE heat-shock gene was found by sequencing in the genome of the methanogenic archaeon Methanosarcina mazie S-6. It is the first example of grpE from the phylogenetic domain Archea. Since the other seven sequenced homologs are from the domain Bacteria, it may be concluded that grpE appeared early in evolution, before the two domains separated. The archaeal grpE is located in the dnaK locus, 431 base-pairs upstream of dnaK, which is followed downstream by the dnaJ gene. The organization of these three genes is known for Bacillus subtilis, Clostridium acetobutylicum , Borrelia burgdorferi and Mycobacterium tuberculosis . The archaeal locus organization, grpE-dnaK-dnaJ , is similar to that of the former three bacteria, but different from that of M. tuberculosis. This, and sequence homologies, suggest that the M. tuberculosis GrpE belongs, together with the Streptomyces coelicolor homolog, to a subgroup of the GrpE proteins. The M. mazie grpE gene encodes a protein of 209 amino acid residues. The deduced amino acid sequence shows 28?2 to 34?6% identities, and 50?93 to 58?9 similarities (identities plus conservative substitutions) with the other six complete GrpE sequences available. These percentages fall within the range observed for the other GrpEs. Two regions in the second and fourth quarters of the GrpE molecule show higher homology, particularly in three stretches of nine, six and nine amino acid residues, respectively. The archaeal gene uses all codons but there whereas the bacterial homologs lack higher numbers of codons. The M. mazie grpE respond to heat-shock by increasing transcription, in a manner similar to that of the nearby heat-shock gene dnaK .

YGE1 is a yeast homologue of Escherichia coli grpE and is required for maintenance of mitochondrial functions

The grpE gene is a heat shock gene of Escherichia coli whose product functions as a chaperone to (re)fold proteins. We found a yeast homologue of grpE and designated it YGE1. YGE1 can replace grpE in E coli, indicating that YGE1 is a functional homologue of grpE. Deletion of YGE1 is lethal. During depletion of the Ygel product, mitochondria are sequestered in mother cells thereby accumulating cells without mitochondria, suggesting that Ygel protein plays a pivotal role in maintaining mitochondrial functions.

Isolation of dnaJ, dnaK, and grpE homologues from Borrelia burgdorferi and complementation of Escherichia coli mutants

The heat-shock proteins DnaJ, DnaK, and GrpE are involved in the replication of various species of DNA in Escherichia coli, in addition to their roles in other processes, including protein disaggregation and export. We have cloned the Borrelia burgdorferi homologues of these genes. DNA sequence analysis revealed an open reading frame encoding a protein that is 62% identical to the E. coli DnaK protein. Genes homologous to the E. coli grpE and dnaJ genes, encoding products 28% and 39% identical to their homologues, are located up- and downstream, respectively, of the B. burgdorferi dnaK gene. No obvious promoters were detected in the sequenced DNA, although a potential transcription terminator was found downstream of the dnaJ gene, so these three genes may form an operon, perhaps with a fourth gene located upstream of the grpE gene. The grpE homologue complemented an E. coli grpE mutant and the dnaJ homologue complemented an E. coli dnaJ mutant, whereas the B. burgdorferi dnaK gene did not complement dnaK mutants.

DnaK, DnaJ, and GrpE are required for flagellum synthesis in Escherichia coli.

The DnaK, DnaJ, and GrpE heat shock proteins are required for motility of Escherichia coli. Cells deleted for dnaK or dnaJ, or with some mutations in the dnaK or grpE gene, are nonmotile, lack flagella, exhibit a 10- to 20-fold decrease in the rate of synthesis of flagellin, and show reduced rates of transcription of both the flhD master operon (encoding FlhD and FlhC) and the fliA operon (encoding sigma F). Genetic studies suggest that DnaK and DnaJ define a regulatory pathway affecting flhD and fliA synthesis that is independent of cyclic AMP-catabolite gene activator protein or the chemotaxis system.

The essential Escherichia coli msgB gene, a multicopy suppressor of a temperature-sensitive allele of the heat shock gene grpE, is identical to dapE.

The grpE gene product is one of three Escherichia coli heat shock proteins (DnaK, DnaJ, and GrpE) that are essential for both bacteriophage lambda DNA replication and bacterial growth at all temperatures. In an effort to determine the role of GrpE and to identify other factors that it may interact with, we isolated multicopy suppressors of the grpE280 point mutation, as judged by their ability to reverse the temperature-sensitive phenotype of grpE280. Here we report the characterization of one of them, designated msgB. The msgB gene maps at approximately 53 min on the E. coli chromosome. The minimal gene possesses an open reading frame that encodes a protein with a predicted size of 41,269 M(r). This open reading frame was confirmed the correct one by direct amino-terminal sequence analysis of the overproduced msgB gene product. Genetic experiments demonstrated that msgB is essential for E. coli growth in the temperature range of 22 to 37 degrees C. Through a sequence homology search, MsgB was shown to be identical to N-succinyl-L-diaminopimelic acid desuccinylase (the dapE gene product), which participates in the diaminopimelic acid-lysine pathway involved in cell wall biosynthesis. Consistent with this finding, the msgB null allele mutant is viable only when the growth medium is supplemented with diaminopimelic acid. These results suggest that GrpE may have a previously unsuspected function(s) in cell wall biosynthesis in E. coli.

Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.

The products of the Escherichia coli dnaK, dnaJ, and grpE heat shock genes have been previously shown to be essential for bacteriophage lambda DNA replication at all temperatures and for bacterial survival under certain conditions. DnaK, the bacterial heat shock protein hsp70 analogue and putative chaperonin, possesses a weak ATPase activity. Previous work has shown that ATP hydrolysis allows the release of various polypeptides complexed with DnaK. Here we demonstrate that the ATPase activity of DnaK can be greatly stimulated, up to 50-fold, in the simultaneous presence of the DnaJ and GrpE heat shock proteins. The presence of either DnaJ or GrpE alone results in a slight stimulation of the ATPase activity of DnaK. The action of the DnaJ and GrpE proteins may be sequential, since the presence of DnaJ alone leads to an acceleration in the rate of hydrolysis of the DnaK-bound ATP. The presence of GrpE alone increases the rate of release of bound ATP or ADP without affecting the rate of hydrolysis. The stimulation of the ATPase activity of DnaK may contribute to its more efficient recycling, and it helps explain why mutations in dnaK, dnaJ, or grpE genes often exhibit similar pleiotropic phenotypes.

Heat shock response of murine Chlamydia trachomatis

We have investigated the heat shock response in the mouse pneumonitis strain of Chlamydia trachomatis. The kinetics of the chlamydial heat shock response resembled that of other procaryotes: the induction was rapid, occurring over a 5- to 10-min time period, and was regulated at the level of transcription. Immunoblot analysis and immunoprecipitations with heterologous antisera to the heat shock proteins DnaK and GroEL demonstrated that the rate of synthesis, but not the absolute amount of these two proteins, increased after heat shock. Using a general screen for genes whose mRNAs are induced by heat shock, we identified and cloned two of these. DNA sequence analysis demonstrated that one of the genes is a homolog of dnaK. Further sequence analysis of the region upstream of the dnaK gene revealed that the chlamydial homolog of the grpE gene is located just adjacent to the dnaK gene. The second locus encoded three potential nonoverlapping open reading frames. One of the open reading frames was 52% homologous to the ribosomal protein S18 of Escherichia coli and thus presumably encodes the chlamydial homolog. Interestingly, this ribosomal protein is not known to be induced by heat shock in E. coli. S1 nuclease and primer extension analyses located the start site of the dnaK transcript to the last nucleotide of the grpE coding sequence, suggesting that these two genes, although tandemly arranged, are transcribed separately. No promoter sequences resembling the E. coli consensus heat shock promoter could be identified upstream of either the C. trachomatis dnaK, grpE, or S18 gene. The induction of the dnaK and S18 mRNAs by heat shock occurred at a transcriptional level; their induction could be blocked by rifampin. The mechanisms of induction for these two loci were not the same, however; they were differentially sensitive to chloramphenicol. Whereas the induction of dnaK mRNA required de novo protein synthesis, the induction of the S18 mRNA did not. Thus, C. trachomatis utilizes at least two different pathways to induce the transcription of mRNAs encoding proteins induced in the heat shock response.

Isolation and characterization of dnaJ null mutants of Escherichia coli

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.

A grpE mutant of Escherichia coli is more resistant to heat than the wild-type

The grpE gene of Escherichia coli is essential for bacteriophage lambda DNA replication and is also necessary for host RNA and DNA synthesis at high temperature. A grpE mutant of E. coli was found to be substantially more resistant to 50 degrees C heat treatment than the wild-type. Upon receiving a 42 degrees C heat shock for 15 min, both the wild-type and the grpE mutant became more resistant to heat (i.e. they became thermotolerant). A grpE+ revertant behaved similarly to the wild-type in that it was more sensitive to heat than grpE cells. In addition, grpE cells had the same H2O2 and UV sensitivity as the wild-type. This implies that the conditions for which a grpE mutation is beneficial are unique to heat exposure and are not caused by H2O2 or UV exposure. Furthermore, synthesis of heat-shock proteins occurred sooner in the grpE mutant than in the wild-type, indicating that the grpE gene of E. coli may influence the regulation of the heat-shock response.