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1.
The essential Escherichia coli msgB gene, a multicopy suppressor of a temperature-sensitive allele of the heat shock gene grpE, is identical to dapE.
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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. 相似文献
2.
Identification and characterization of a new Escherichia coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation. 总被引:15,自引:12,他引:3
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We report the isolation and characterization of a previously unidentified Escherichia coli gene that suppresses the temperature-sensitive growth and filamentation of a dnaK deletion mutant strain. Introduction of a multicopy plasmid carrying this wild-type gene into a dnaK deletion mutant strain rescued the temperature-sensitive growth of the dnaK deletion mutant strain at 40.5 degrees C and the filamentation, fully at 37 degrees C and partially at 40.5 degrees C. However, the inability of dnaK mutant cells to support bacteriophage lambda growth was not suppressed. This gene was also able to suppress the temperature-sensitive growth of a grpE280 mutant strain at 41 degrees C. Filamentation of the grpE280 mutant strain was suppressed at 37 degrees C but not at 41 degrees C. The dnaK suppressor gene, designated dksA, maps near the mrcB gene (3.7 min on the E. coli chromosome). DNA sequence analysis and in vivo experiments showed that dksA encodes a 17,500-Mr polypeptide. Gene disruption experiments indicated that dksA is not an essential gene. 相似文献
3.
Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro. 总被引:22,自引:15,他引:7
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Previous studies have demonstrated that the Escherichia coli dnaK and grpE genes code for heat shock proteins. Both the Dnak and GrpE proteins are necessary for bacteriophage lambda DNA replication and for E. coli growth at all temperatures. Through a series of genetic and biochemical experiments, we have shown that these heat shock proteins functionally interact both in vivo and in vitro. The genetic evidence is based on the isolation of mutations in the dnaK gene, such as dnaK9 and dnaK90, which suppress the Tr- phenotype of bacteria carrying the grpE280 mutation. Coimmunoprecipitation of DnaK+ and GrpE+ proteins from cell lysates with anti-DnaK antibodies demonstrated their interaction in vitro. In addition, the DnaK756 and GrpE280 mutant proteins did not coimmunoprecipitate efficiently with the GrpE+ and DnaK+ proteins, respectively, suggesting that interaction between the DnaK and GrpE proteins is necessary for E. coli growth, at least at temperatures above 43 degrees C. Using this assay, we found that one of the dnaK suppressor mutations, dnaK9, reinstated a protein-protein interaction between the suppressor DnaK9 and GrpE280 proteins. 相似文献
4.
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. 相似文献
5.
Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both lambda DNA replication at all temperatures and host growth at high temperature. 总被引:10,自引:20,他引:10
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We have identified the grpE gene product as the B25.3 heat shock protein of Escherichia coli on the following evidence: (i) a protein similar in size and isoelectric point to B25.3 was induced after infection of UV-irradiated bacteria by lambda grpE+ transducing phage, (ii) mutant phage lambda grpE40, isolated by its inability to propagate on grpE280 bacteria, failed to induce the synthesis of the B25.3 protein, and (iii) lambda grpE+ revertants, derived from phage grpE40 as able to propagate on grpE280 bacteria, simultaneously recovered the ability to induce synthesis of the B25.3 protein. In addition, we show that E. coli bacteria carrying the grpE280 mutation are temperature sensitive for bacterial growth at 43.5 degrees C. Through transductional analysis and temperature reversion experiments, it was demonstrated that the grpE280 mutation is responsible for both the inability of lambda to replicate at any temperature tested and the lack of colony formation at high temperature. At the nonpermissive temperature the rates of synthesis of DNA and RNA were reduced in grpE280 bacteria. 相似文献
6.
J M Delaney 《Journal of general microbiology》1990,136(5):797-801
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. 相似文献
7.
Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes. Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception. Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues. The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function. The M. mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2. Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M. mazei DnaK. The M. mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison. However, in vivo analyses indicate that there are also significant differences. The M. mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M. mazei dnaK gene. Thus, while the data from in vitro tests demonstrate functional similarities between the M. mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ. 相似文献
8.
Ribonuclease D is not essential for the normal growth of Escherichia coli or bacteriophage T4 or for the biosynthesis of a T4 suppressor tRNA 总被引:6,自引:0,他引:6
Transposon Tn10-mediated rearrangement was used to isolate a strain of Escherichia coli carrying a deletion in the rnd region which is known to encode the structural gene for the putative 3' tRNA processing nuclease, RNase D. Genetic analysis indicated that about 0.4-0.5 min of the chromosome in the 39.5-40.0 min region was deleted. The mutant strain was devoid of RNase D activity, but other RNase activities were unaffected. The viability of the mutant strain and its normal growth characteristics indicate that RNase D is not essential for E. coli survival. The normal plating efficiency in this mutant host of wild type T4 and a T4 psu1+-amber double mutant indicates that RNase D is also not required for T4 growth or psu1+-tRNA processing. The implications of these findings for the role of RNase D in bacterial and bacteriophage tRNA metabolism, and the possible involvement of alternative enzymes, are discussed. 相似文献
9.
10.
We have constructed four deletion derivatives of the cloned dnaK gene. Plasmid pDD1, in which the last 10 amino acids of the DnaK protein have been replaced by three different amino acids derived from the pBR322 vector, was as effective as plasmid pKP31, from which it was derived, in restoring the ability of a dnaK null mutant, Escherichia coli BB1553, to plate lambda phage and to grow at high temperatures. The other three mutations, involving much larger deletions of the dnaK gene, did not restore the ability to plate lambda phage or the ability to grow at high temperatures. Plasmid pKUC2, which contains the whole dnaK gene and its promoters, was capable of restoring the ability of E. coli BB1553 to plate lambda phage but, surprisingly, it did not restore the ability to grow at high temperatures, even though it was shown that the DnaK protein was efficiently expressed in these cultures. By transposon mutagenesis and sub-cloning, we have shown the presence of a second gene in plasmid pKP31 which is required for high-temperature growth of E. coli BB1553. This gene, which we call htg A, is presumably also defective in the dnaK null mutant E. coli BB1553. We have also demonstrated that the inability of E. coli K756 to grow above 43.5 degrees C is complemented by sub-clones which contain the htg A gene, but not by plasmid pKUC2. 相似文献
11.
DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
The growth of Escherichia coli cells is impaired at temperatures below 21 degrees C and stops at 7.5 degrees C; however, growth of a transgenic strain producing the cold-adapted chaperones Cpn60 and Cpn10 from the psychrophilic bacterium Oleispira antarctica is good at low temperatures. The E. coli cpn(+) transgene offers a novel opportunity for examining the essential protein for cell viability at low temperatures. By screening a large-scale protein map (proteome) of cells of K-12 and its Cpn(+) transgene incubated at 4 degrees C, we identified 22 housekeeping proteins involved in systems failure of E. coli when confronted with low temperature. Through co-immunoprecipitation of Cpn60, Northern blot, and in vitro refolding, we systematically identified that protein-chaperone interactions are key determinants of their protein functions at low temperatures. Furthermore, chromosomal gene deletion experiments suggest that the mechanism of cold-induced systems failure in E. coli is cold-induced inactivation of the GroELS chaperonins and the resulting failure to refold cold-inactivated Dps, ClpB, DnaK and RpsB proteins. These findings: (1) indicate the potential importance of chaperones in cold sensitivity, cold adaptation and cold tolerance in cellular systems, and (2) suggest the identity of a few key cold-sensitive chaperone-interacting proteins that get inactivated and ultimately cause systems failure in E. coli cells at low temperatures. 相似文献
13.
14.
Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media. 总被引:15,自引:8,他引:7
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M Karow O Fayet A Cegielska T Ziegelhoffer C Georgopoulos 《Journal of bacteriology》1991,173(2):741-750
We have identified and studied the htrB gene of Escherichia coli. Insertional inactivation of the htrB gene leads to bacterial death at temperatures above 33 degrees C. The mutant bacterial phenotype at nonpermissive temperatures includes an arrest of cell division followed by the formation of bulges or filaments. The htrB+ gene has been cloned by complementation and shown to reside at 23.4 min on the E. coli genetic map, the relative order of the neighboring loci being mboA-htrB-pyrC. The htrB gene is transcribed in a counterclockwise fashion, relative to the E. coli genetic map, and its product has been identified as a membrane-associated protein of 35,000 Da. Growth experiments in minimal media indicate that the HtrB function becomes dispensable at low growth rates. 相似文献
15.
Role of heat shock protein DnaK in osmotic adaptation of Escherichia coli. 总被引:7,自引:3,他引:4
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Escherichia coli can adapt and recover growth at high osmolarity. Adaptation requires the deplasmolysis of cells previously plasmolyzed by the fast efflux of water promoted by osmotic upshift. Deplasmolysis is essentially ensured by a net osmo-dependent influx of K+. The cellular content of the heat shock protein DnaK is increased in response to osmotic upshift and does not decrease as long as osmolarity is high. The dnaK756(Ts) mutant, which fails to deplasmolyze and recover growth, does not take up K+ at high osmolarity; DnaK protein is required directly or indirectly for the maintenance of K+ transport at high osmolarity. The temperature-sensitive mutations dnaJ259 and grpE280 do not affect the osmoadaptation of E. coli at 30 degrees C. 相似文献
16.
Escherichia coli DnaK and rat Hsc70 are members of the highly conserved 70-kDa heat shock protein (Hsp70) family that show strong sequence and structure similarities and comparable functional properties in terms of interactions with peptides and unfolded proteins and cooperation with cochaperones. We show here that, while the DnaK protein is, as expected, able to complement an E. coli dnaK mutant strain for growth at high temperatures and lambda phage propagation, Hsc70 protein is not. However, an Hsc70 in which the peptide-binding domain has been replaced by that of DnaK is able to complement this strain for both phenotypes, suggesting that the peptide-binding domain of DnaK is essential to fulfill the specific functions of this protein necessary for growth at high temperatures and for lambda phage replication. The implications of these findings on the functional specificities of the Hsp70s and the role of protein-protein interactions in the DnaK chaperone system are discussed. 相似文献
17.
To investigate the function of spot 42 RNA, a small RNA of Escherichia coli, we constructed a strain in which spf, the structural gene for this RNA, is deleted. We achieved this by using a delta att phage lambda carrying a DNA fragment spanning the spf region but with a precise deletion of spf. By integration of this phage at the spf locus and by its subsequent excision, we were able to cross the spf deletion onto the bacterial chromosome. The fact that such a deletion could be obtained indicated that spf is not an essential gene. We did not observe any major defect in delta spf cells, although in one strain background the deletion caused a slight growth impairment. 相似文献
18.
A mitochondrial homolog of bacterial GrpE interacts with mitochondrial hsp70 and is essential for viability. 总被引:21,自引:8,他引:13
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L Bolliger O Deloche B S Glick C Georgopoulos P Jen? N Kronidou M Horst N Morishima G Schatz 《The EMBO journal》1994,13(8):1998-2006
Mitochondrial hsp70 (mhsp70) is located in the matrix and an essential component of the mitochondrial protein import system. To study the function of mhsp70 and to identify possible partner proteins we constructed a yeast strain in which all mhsp70 molecules carry a C-terminal hexa-histidine tag. The tagged mhsp70 appears to be functional in vivo. When an ATP depleted mitochondrial extract was incubated with a nickel-derivatized affinity resin, the resin bound not only mhsp70, but also a 23 kDa protein. This protein was dissociated from mhsp70 by ATP. ADP and GTP were much less effective in promoting dissociation whereas CTP and TTP were inactive. We cloned the gene encoding the 23 kDa protein. This gene, termed GRPE, encodes a 228 residue protein, whose sequence closely resembles that of the bacterial GrpE protein. Microsequencing the purified 23 kDa protein established it as the product of the yeast GRPE gene. Yeast GrpEp is made as a precursor that is cleaved upon import into isolated mitochondria. GrpEp is essential for viability. We suggest that this protein interacts with mhsp70 in a manner analogous to that of GrpE with DnaK of E.coli. 相似文献
19.
The DnaK chaperone is necessary for alpha-complementation of beta-galactosidase in Escherichia coli
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We show here the involvement of the molecular chaperone DnaK from Escherichia coli in the in vivo alpha-complementation of the beta-galactosidase. In the dnaK756(Ts) mutant, alpha-complementation occurs when the organisms are grown at 30 degrees C but not at 37 or 40 degrees C, although these temperatures are permissive for bacterial growth. Plasmid-driven expression of wild-type dnaK restores the alpha-complementation in the mutant but also stimulates it in a dnaK(+) strain. In a mutant which contains a disrupted dnaK gene (DeltadnaK52::Cm(r)), alpha-complementation is also impaired, even at 30 degrees C. This observation provides an easy and original phenotype to detect subtle functional changes in a protein such as the DnaK756 chaperone, within the physiologically relevant temperature. 相似文献
20.
rpoZ, encoding the omega subunit of Escherichia coli RNA polymerase, is in the same operon as spoT 总被引:6,自引:2,他引:4
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Highly purified Escherichia coli RNA polymerase contains a small subunit termed omega. This subunit consists of 91 amino acids with a molecular weight of 10,105. We previously reported the cloning and sequencing of the gene encoding omega, which we call rpoZ (D. R. Gentry and R. R. Burgess, Gene 48:33-40, 1986). We constructed an rpoZ insertion mutation by placing a kanamycin resistance cassette into the coding region of the rpoZ gene. Purified RNA polymerase from strains carrying this mutation lacked detectable omega. We found that the insertion mutation conferred a slow-growth phenotype when introduced into most strains. We mapped the position of rpoZ on the E. coli chromosome by genetic techniques and by examining the restriction map of the whole chromosome and found that rpoZ maps around 82 min, very close to spoT. We determined that the slow-growth phenotype of the insertion mutant is suppressed in relA mutants and that the rpoZ insertion results in a classical SpoT- phenotype. This finding strongly suggests that rpoZ is upstream of spoT in the same operon and that the slow-growth phenotype elicited by the insertion mutation is due to polarity on spoT. 相似文献