Cloning and complementation analysis of the Escherichia coli gpr locus, influencing DNA replication of certain lamdoid phages]

Escherichia coli DNA fragments which suppressed gpr27 and gpr2 mutations in the earlier proposed gprA and gprB genes, respectively, were cloned within phage vectors. Mutations gpr2 and gpr27 restrict DNA replication of some lambdoid phages and are located in the region of dnaK, J genes. DNA-DNA hybridization showed that the cloned fragments correspond to the region where gpr mutations were genetically mapped and, in some cases, do not include dnaK, J genes. These results provide the evidence that gprA and gprB genes may be physically separated from dnaK, J genes.     

Identification of the C. coli dnaK (groPC756) gene product.

Summary The E. coli dnaK (groPC756) gene product is essential for bacteriophage DNA replication. Bacterial DNA segments carrying this gene have been cloned onto a bacteriophage vector. The product of the dnaK gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells. The dnaK gene codes for a polypeptide with an apparent molecular weight of 93,000-Mr. Transducing phages carrying amber mutations in the dnaK gene fail to induce the synthesis of the 93,000-Mr polypeptide chain upon infection of sup ⁺ bacteria, but do so upon infection of supF bacteria. E. coli carrying the dnaK756 mutation are, in addition, temperature sensitive for growth at 43° C. It is shown that the dnaK756 mutation results in an overproduction of the dnaK gene product at that temperature.     

The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells

The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins. In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C. In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min). This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ. The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent. It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins. Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates. The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system.     

Purification and properties of the dnaJ replication protein of Escherichia coli

The Escherichia coli dnaJ gene was originally discovered because mutations in it blocked bacteriophage lambda DNA replication. Some of these mutations were subsequently shown to interfere with bacterial growth at high temperature, suggesting that dnaJ is an essential protein for the host as well. The first step in purifying the dnaJ protein was to overproduce it at least 50-fold by subcloning its gene into the pMOB45 runaway plasmid. The second step was the development of an in vitro system to assay for its activity. A Fraction II extract from dnaJ259 mutant bacteria was shown to be unable to replicate lambda dv DNA unless supplemented with an exogenous source of wild-type dnaJ protein. Using this complementation assay we purified the dnaJ protein to homogeneity from the membrane fraction of an overproducing strain of bacteria. The purified dnaJ protein was shown to be a basic (pI 8.5), yet hydrophobic, protein of Mr 37,000 and 76,000 under denaturing and native conditions, respectively, and to exhibit affinity for both single- and double-stranded DNA. Using a partially purified lambda dv replication system dependent on the presence of the lambda O and P initiator proteins and at least the host dnaB, dnaG, dnaJ, dnaK, single-stranded DNA-binding protein, gyrase, RNA polymerase holoenzyme, and DNA polymerase III holoenzyme, we have shown that the dnaJ protein is required at a very early step in the DNA replication process.     

Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product

The dnaJ and dnaK genes are essential for replication of Escherichia coli DNA, and they constitute an operon, dnaJ being downstream from dnaK. The amount of the dnaJ protein in E. coli is substantially less than that of the dnaK protein, which is produced abundantly. In order to construct a system that over-produces the dnaJ protein, we started our study by determining the DNA sequence of the entire dnaJ gene, and an operon fusion was constructed by inserting the gene downstream of the lambda PL promoter of an expression vector plasmid, pPL-lambda. Cells containing the recombinant plasmid produced dnaJ protein amounting to 2% of the total cellular protein when cells were induced. The overproduced protein was purified, and Edman degradation of the protein indicated that the NH2-terminal methionine was found to be processed. From the DNA sequence of the dnaJ gene, the processed gene product is composed of 375 amino acid residues, and its molecular weight is calculated to be 40,975.     

Phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase by the gene products of dnaK and dnaJ in Escherichia coli K-12 cells

In Escherichia coli K-12, the heat shock protein DnaK and DnaJ participate in phosphorylation of both glutaminyl-tRNA synthetase and threonyl-tRNA synthetase since when cellular proteins extracted from the dnaK7(Ts), dnaK756(Ts) and dnaJ259(Ts) mutant cells labeled with 32Pi at 42 degrees C were analyzed by two-dimensional gel electrophoresis, no phosphorylation of glutaminyl-tRNA synthetase and threonyl-tRNA synthetase was observed while phosphorylation of both aminoacyl-tRNA synthetases was detected in the samples extracted from wild-type cells.     

Escherichia coli DnaK and GrpE heat shock proteins interact both in vivo and in vitro.

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.     

Role of heat shock protein DnaK in osmotic adaptation of Escherichia coli.

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.     

Involvement of the chaperonin dnaK in the rapid degradation of a mutant protein in Escherichia coli.

The ability of Escherichia coli rapidly to degrade abnormal proteins is inhibited by mutations affecting any of several heat shock proteins (hsps). We therefore tested whether a short-lived mutant protein might become associated with hsps as part of its degradation. At 30 degrees C, the non-secreted mutant form of alkaline phosphatase, phoA61, is relatively stable, and very little phoA61 is found associated with the hsp dnaK. However, raising the temperature to 37 degrees C or 41 degrees C stimulated the degradation of this protein, and up to 30% of cellular phoA61 became associated with dnaK, as shown by immunoprecipitation and Western blot analysis. Also found in complexes with phoA61 were the hsps, protease La and grpE (but no groEL, or groES). The rapid degradation of phoA61 at 37 degrees C and 41 degrees C is in part by protease La, since it decreased by 50% in lon mutants. This process also requires dnaK, since deletion of this gene prevented phoA61 degradation almost completely (unless a wild-type dnaK gene was introduced). In contrast, the missense mutation, dnaK756, enhanced phoA61 degradation. The dnaK756 protein also was associated with phoA61, but this complex, unlike that containing wild-type dnaK could not be dissociated by ATP addition. Furthermore, in a grpE mutant, the degradation of phoA61 and the amount associated with dnaK increased, while in a dnaJ mutant, phoA61 degradation and its association with dnaK decreased. Thus, complex formation with dnaK appears essential for phoA61 degradation by protease La and some other cell proteases, and a failure of the dnaK to dissociate normally may accelerate proteolytic attack.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene structure and transcriptional regulation of <Emphasis Type="Italic">dnaK</Emphasis> and <Emphasis Type="Italic">dnaJ</Emphasis> genes from a psychrophilic bacterium, <Emphasis Type="Italic">Colwellia maris</Emphasis>

The dnaK and dnaJ genes, encoding heat shock proteins, were cloned from a psychrophilic bacterium, Colwellia maris. Significant homology was evident comparing DnaK and DnaJ of the psychrophilile with the counterparts of mesophilic and thermophilic bacteria. In the DnaJ protein, three conserved regions of the Hsp40 family were observed. A putative promoter similar to the sigma32 consensus sequence was found upstream of the dnaK gene. The G+C content in the 5'-untranslated region of the dnaK gene was much lower than that in the corresponding region of mesophilic bacteria. Northern-blot analysis and primer-extension analysis showed that both genes were transcribed separately as monocistronic mRNAs. Following several temperature upshifts from 10 to 26 degrees C, maximum induction of the dnaK and dnaJ mRNAs was detected at 20 degrees C, suggesting that this temperature induces the heat shock response in this bacterium. In addition, the level of the induction of the dnaJ gene was much lower than that of the dnaK gene. These findings together revealed several specific features of the heat shock response at a relatively low temperature in psychrophiles.     

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.     

Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis

The employment of a set of truncated dnaK peptides produced by deletion and insertion mutations in the Escherichia coli dnaK gene allowed us to define regions of the dnaK protein which are involved in particular enzymatic functions. The results obtained suggest that the dnaK polypeptide is organized into at least two distinct functional domains. The highly conserved amino-terminal portion is required for the ATPase activity. The carboxyl-terminal portion, characterized by relatively low similarity among species, is responsible for the autophosphorylating activity. The mutant dnaK protein C[74], which lacks amino acid sequences at the extreme carboxyl-terminal portion of the protein, retains both the ATPase and the autophosphorylating activities. The results obtained with the full-length (70-kDa) dnaK756 protein suggest that the thermolabile defect of the dnaK756 mutation affects directly or indirectly the ATPase active site of the enzyme. The autophosphorylating activity of the dnaK+, dnaK756, and C[74] polypeptides was activated at least 10-fold by the addition of CaCl2.     

Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication.

Low-copy-number plasmids, such as P1 prophage and the fertility factor F, require a plasmid-encoded replication protein and several host products for replication. Stable maintenance also depends on active partitioning of plasmids into daughter cells. Mini-P1 par+ and par plasmids were found to be destabilized by mutations in the dnaJ, dnaK, and grpE genes of Escherichia coli. The transformation efficiency and stability of mini-F plasmids were also reduced in the mutant strains. These results indicate that heat shock proteins DnaJ, DnaK, and GrpE play roles in the replication of plasmid P1 and probably also in of F.     

Participation of the Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in autorepression of the P1 plasmid repA promoter

The replicon of the low copy number plasmid P1 uses the three Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE for the efficient initiation of its DNA replication. The only P1-encoded protein required for plasmid replication is the initiator, RepA. Binding of RepA to the origin also represses the promoter for the repA gene, which is located within the origin. We found that repression is incomplete in E. coli strains with mutations in the dnaJ, dnaK, or grpE genes. Since there is no decrease in RepA concentration in the mutant strains, the mutations are likely to affect the protein-DNA or protein-protein reactions required for repression, thereby decreasing RepA binding at its promoter. We also showed that the deficit in repression can be overcome by providing excess RepA, implying that the mechanism of repression is not altered in the mutant strains. Since repression requires RepA binding to the origin, a binding deficit might account for the replication defect in the heat shock mutants.