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.     

Characterization of Campylobacter jejuni RacRS reveals roles in the heat shock response, motility, and maintenance of cell length homogeneity

Campylobacter jejuni commensally colonizes the cecum of birds. The RacR (reduced ability to colonize) response regulator was previously shown to be important in avian colonization. To explore the means by which RacR and its cognate sensor kinase RacS may modulate C. jejuni physiology and colonization, ΔracR and ΔracS mutations were constructed in the invasive, virulent strain 81-176, and extensive phenotypic analyses were undertaken. Both the ΔracR and ΔracS mutants exhibited a ~100-fold defect in chick colonization despite no (ΔracS) or minimal (ΔracR) growth defects at 42 °C, the avian body temperature. Each mutant was defective for colony formation at 44°C and in the presence of 0.8% NaCl, both of which are stresses associated with the heat shock response. Promoter-reporter and real-time quantitative PCR (RT-qPCR) analyses revealed that RacR activates racRS and represses dnaJ. Although disregulation of several other heat shock genes was not observed at 38°C, the ΔracR and ΔracS mutants exhibited diminished upregulation of these genes upon a rapid temperature upshift. Furthermore, the ΔracR and ΔracS mutants displayed increased length heterogeneity during exponential growth, with a high proportion of filamented bacteria. Filamented bacteria had reduced swimming speed and were defective for invasion of Caco-2 epithelial cells. Soft-agar studies also revealed that the loss of racR or racS resulted in whole-population motility defects in viscous medium. These findings reveal new roles for RacRS in C. jejuni physiology, each of which is likely important during colonization of the avian host.     

Biosynthesis and secretion of several enzymes in Escherichia coli dnaK and dnaJ mutants

Escherichia coli null dnaJ and dnaKdnaJ mutants were defective in the biosynthesis and secretion of several enzymes. The synthesis of beta-galactosidase induced in delta dnaJ and delta dnaKdnaJ mutants was abolished at 42 degrees C and significantly decreased at 30 and 37 degrees C. The activity of alkaline phosphatase in the periplasm in both mutant strains at high temperature was lower than in the wild-type strain. The synthesis of b-type cytochromes was defective in two deletion mutants while the synthesis of nitrate reductase-A at 42 degrees C was influenced by dnaK mutation only. The lack of DnaK and DnaJ does not impair the activity of catechol 2,3-dioxygenase irrespective of growth temperature.     

Physiological consequences of mutations in Escherichia coli heat shock dnaK and dnaJ genes.

Mutations in the heat shock genes, dnaK and dnaJ, cause severe defects of several cellular functions. Null dnaJ and dnaKdnaJ mutations can be transduced in a restricted range of temperature. The efficiency of transformation with three unrelated plasmids, viz pACYC184, pBR322 and pSC101, is two times lower in dnaK mutants while the dnaJ mutant is characterized by slightly impaired transformation with pSC101 only. The lack of DnaJ function negatively influences the stability of pSC101 at 42 degrees C, and this plasmid cannot be stably maintained at 30 degrees C in the delta dnaKdnaJ mutant. The double deletion mutant, delta dbaKdnaJ, is characterized by impaired osmoadaptation. The galactokinase content is lower in both mutants tested compared with wild-type strains even at 30 degrees C. The efficient complementation of some of these defects by the wild-type alleles present on low-copy number plasmid was achieved.     

The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures.

The products of the groES and groEL genes of Escherichia coli, constituting the groE operon, are known to be required for growth at high temperature (42 degrees C) and are members of the heat shock regulon. Using a genetic approach, we examined the requirement for these gene products for bacterial growth at low temperature (17 to 30 degrees C). To do this, we constructed various groES groEL heterodiploid derivative strains. By inactivating one of the groE operons by a polar insertion, it was shown by bacteriophage P1 transduction that at least one of the groE genes was essential for growth at low temperature. Further P1 transduction experiments with strains that were heterodiploid for only one of the groE genes demonstrated that both groE gene products were required for growth at low temperature, which suggested a fundamental role for the groE proteins in E. coli growth and physiology.     

Lysis of Escherichia coli by the bacteriophage phi X174 E protein: inhibition of lysis by heat shock proteins.

Lysis of Escherichia coli by the cloned E protein of bacteriophage phi X174 was more rapid than expected when bacteria were shifted from 30 to 42 degrees C at the time of E induction. Since such treatment also induces the heat shock response, we investigated the effect of heat shock proteins on lysis. An rpoH mutant was more sensitive to lysis by E, but a secondary suppressor mutation restored lysis resistance to parental levels, which suggests that the sigma 32 subunit itself did not directly increase lysis resistance. At 30 degrees C, mutants in five heat shock genes (dnaK, dnaJ, groEL, groES, and grpE) were more sensitive to lysis than were their wild-type parents. The magnitude of lysis sensitivity varied with mutation and strain background, with dnaK, dnaJ, and groES mutants consistently exhibiting the greatest sensitivities. Extended protection against lysis occurred when overproduction of heat shock proteins was induced artificially in cells that contained a plasmid with the rpoH+ gene under control of the tac promoter. This protective effect was completely abolished by mutations in dnaK, dnaJ, or groES but not by grpE or groEL mutations. Altered membrane behavior probably explains the contradiction whereby an actual temperature shift sensitized cells to lysis, but production of heat shock proteins exhibited protective effects. The results demonstrate that E-induced lysis can be divided into two distinct operations which may now be studied separately. They also emphasize a role for heat shock proteins under non-heat-shock conditions and suggest cautious interpretation of lysis phenomena in systems where E protein production is under control of a temperature-sensitive repressor.     

Chromosomal rearrangements by an IS2 insertion in phage Mu-1

We have isolated and characterized a mutant of temperate phage Mu-1 carrying an IS2 insertion in the middle of its β region. This mutant gives rise spontaneously to secondary mutants which have deletions of different sizes adjacent to IS2. One particular derivative however, was found to have acquired an additional insertion sequence adjacent to IS2. This derivative gave rise to tertiary mutants carryinh a deletion next to the tandem insertion. The tandem insertion was located at the same place in the Mu β region as another 2.6 kb insertion independently isolated by Chow et al. (1977) and was found to be homologous to that insertion. The properties of this particular secondary mutant show that Mu phage particles lacking their S end are defective for growth and lysogenisation.     

DnaK mutants defective in ATPase activity are defective in negative regulation of the heat shock response: expression of mutant DnaK proteins results in filamentation.

Site-directed mutagenesis has previously been used to construct Escherichia coli dnaK mutants encoding proteins that are altered at the site of in vitro phosphorylation (J. S. McCarty and G. C. Walker, Proc. Natl. Acad. Sci. USA 88:9513-9517, 1991). These mutants are unable to autophosphorylate and are severely defective in ATP hydrolysis. These mutant dnaK genes were placed under the control of the lac promoter and were found not to complement the deficiencies of a delta dnaK mutant in negative regulation of the heat shock response. A decrease in the expression of DnaK and DnaJ below their normal levels at 30 degrees C was found to result in increased expression of GroEL. The implications of these results for DnaK's role in the negative regulation of the heat shock response are discussed. Evidence is also presented indicating the existence of a 70-kDa protein present in a delta dnaK52 mutant that cross-reacts with antibodies raised against DnaK. Derivatives of the dnaK+ E. coli strain MC4100 expressing the mutant DnaK proteins filamented severely at temperatures equal to or greater than 34 degrees C. In the dnaK+ E. coli strain W3110, expression of these mutant proteins caused extreme filamentation even at 30 degrees C. Together with other observations, these results suggest that DnaK may play a direct role in the septation pathway, perhaps via an interaction with FtsZ. Although delta dnaK52 derivatives of strain MC4100 filament extensively, a level of underexpression of DnaK and DnaJ that results in increased expression of the other heat shock proteins did not result in filamentation. The delta dnaK52 allele could be transduced successfully, at temperatures of up to 45 degrees C, into strains carrying a plasmid expressing dnaK+ dnaJ+, although the yield of transductants decreased above 37 degrees C. In contrast, with a strain that did not carry a plasmid expressing dnaK+ dnaJ+, the yield of delta dnaK52 transductants decreased extremely sharply between 39 and 40 degrees C, suggesting that DnaK and DnaJ play one or more roles critical for growth at temperatures of 40 degrees C or greater.     

Involvement of Escherichia coli K-12 DNA polymerase I in the growth of bacteriophage Mu.

We examined several aspects of bacteriophage Mu development in Escherichia coli strains that carry mutations in the polA structural gene for DNA polymerase I (PolI). We found that polA mutants were markedly less efficient than PolI wild-type (PolI+) strains in their capacity to form stable Mu lysogens and to support normal lytic growth of phage Mu. The frequency of lysogenization was determined for polA mutants and their isogenic PolI+ derivatives, with the result that mutants were lysogenized 3 to 8 times less frequently than were PolI+ cells. In one-step growth experiments, we found that phage Mu grew less efficiently in polA cells than in PolI+ cells, as evidenced by a 50 to 100% increase in the latent period and a 20 to 40% decrease in mean burst size in mutant cells. A further difference noted in infected polA strains was a 10-fold reduction in the frequency of Mu-mediated transposition of chromosomal genes to an F plasmid. Pulse labeling and DNA-DNA hybridization assays to measure the rate of phage Mu DNA synthesis after the induction of thermosensitive prophages indicated that phage Mu replication began at about the same time in both polA and PolI+ strains, but proceeded at a slower rate in polA cells. We conclude that PolI is normally involved in the replication and integration of phage Mu. However, since phage Mu does not exhibit an absolute requirement for normal levels of PolI, it appears that residual PolI activity in the mutant strains, other cellular enzymes, or both can partially compensate for the absence of normal PolI activity.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

The dependence of HNO2 mutagenesis in phage T4 on ligase and the lack of dependence of 2AP mutagenesis on repair functions

Summary A temperature sensitive ligase allele of phage T4 reduced or eliminated HNO₂ induced reversion of am mutants. Since at the temperatures used, the ligase mutant is defective in the repair of some types of lethal lesions (i.e., UV, MMS and EMS induced lesions) these results indicate that HNO₂ mutagenesis may occur through a ligase dependent repair pathway. In contrast, 2AP induced mutation was not inhibited by mutants defective in the gene 30 ligase or in genes 32, 39, 41, 44, 45, 46, 47, 49, 52, 56, 58–61 and v. This indicates that 2AP mutagenesis probably does not depend on a repair pathway in phage T4.     

Cloning, characterization, and functional expression in Escherichia coli of chaperonin (groESL) genes from the phototrophic sulfur bacterium Chromatium vinosum.

A recombinant lambda phage which was able to propagate in groE mutants of Escherichia coli was isolated from a Chromatium vinosum genomic DNA library. A 4-kbp SalI DNA fragment, isolated from this phage and subcloned in plasmid vectors, carried the C. vinosum genes that allowed lambda growth in these mutants. Sequencing of this fragment indicated the presence of two open reading frames encoding polypeptides of 97 and 544 amino acids, respectively, which showed high similarity to the molecular chaperones GroES and GroEL, respectively, from several eubacteria and eukaryotic organelles. Expression of the cloned C. vinosum groESL genes in E. coli was greatly enhanced when the cells were transferred to growth temperatures that induce the heat shock response in this host. Coexpression in E. coli of C. vinosum groESL genes and the cloned ribulose bisphosphate carboxylase/oxygenase genes from different phototrophic bacteria resulted in an enhanced assembly of the latter enzymes. These results indicate that the cloned DNA fragment encodes C. vinosum chaperonins, which serve in the assembly process of oligomeric proteins. Phylogenic analysis indicates a close relationship between C. vinosum chaperonins and their homologs present in pathogenic species of the gamma subdivision of the eubacterial division Proteobacteria.     

The Escherichia coli dnaJ mutation affects biosynthesis of specific proteins, including those of the lac operon.

Temperature-sensitive dnaJ mutants of Escherichia coli showed a thermosensitive defect in the synthesis of beta-galactosidase. Synthesis of the lac mRNA was greatly reduced at the restrictive temperature. The mutants were also conditionally defective in the synthesis of a subset of membrane proteins such as succinate dehydrogenase, whereas the synthesis of anthranilate synthetase, encoded by trpED, as well as that of most cellular proteins, was unaffected at the restrictive temperature. The defect was specific for the dnaJ mutants among several dna mutants which are known to be involved in the initiation of DNA synthesis: dnaK, dnaA, and dnaB mutants synthesized each of these proteins normally even at the restrictive temperature. At the restrictive temperature, growth of the dnaJ mutants was arrested at a specific stage of the cell cycle.