Characterization of the Heat Shock Response in Lactococcus lactis subsp. lactis

The heat shock response in Lactococcus lactis subsp. lactis was characterized with respect to synthesis of a unique set of proteins induced by thermal stress. A shift in temperature from 30 to 42°C was sufficient to arrest the growth of L. lactis subsp. lactis, but growth resumed after a shift back to 30°C. Heat shock at 50°C reduced the viable cell population by 10³; however, pretreatment of the cells at 42°C made them more thermoresistant to exposure at 50°C. The enhanced synthesis of approximately 13 proteins was observed in cells labeled with ³⁵S upon heat shock at 42°C. Of these heat shock-induced proteins, two appeared to be homologs of GroEL and DnaK, based on their molecular weights and reactivity with antiserum against the corresponding Escherichia coli proteins. Therefore, we conclude that L. lactis subsp. lactis displays a heat shock response similar to that observed in other mesophilic bacteria.     

Complementation studies of the DnaK–DnaJ–GrpE chaperone machineries from <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>, both in vivo and in vitro

The marine bacterium Vibrio harveyi is a potential indicator organism for evaluating marine environmental pollution. The DnaK–DnaJ–GrpE chaperone machinery of V. harveyi has been studied as a model of response to stress conditions and compared to the Escherichia coli DnaK system. The genes encoding DnaK, DnaJ and GrpE of V. harveyi were cloned into expression vectors and grpE was sequenced. It was found that V. harveyi possesses a unique organization of the hsp gene cluster (grpE–gltP–dnaK–dnaJ), which is present exclusively in marine Vibrio species. In vivo experiments showed that suppression of the E. coli dnaK mutation by V. harveyi DnaK protein was weak or absent, while suppression of the dnaJ and grpE mutations by V. harveyi DnaJ and GrpE proteins was efficient. These results suggest higher species-specificity of the DnaK chaperone than the GrpE and DnaJ cochaperones. Proteins of the DnaK chaperone machinery of V. harveyi were purified to homogeneity and their efficient cooperation with the E. coli chaperones in the luciferase refolding reaction and in stimulation of DnaK ATPase activity was demonstrated. Compared to the E. coli system, the purified DnaK–DnaJ–GrpE system of V. harveyi exhibited about 20% lower chaperoning activity in the luciferase reactivation assay. ATPase activity of V. harveyi DnaK protein was at least twofold higher than that of the E. coli model DnaK but its stimulation by the cochaperones DnaJ and GrpE was significantly (10 times) weaker. These results indicate that, despite their high structural identity (approximately 80%) and similar mechanisms of action, the DnaK chaperones of closely related V. harveyi and E.coli bacteria differ functionally.     

Roles of DnaK and RpoS in Starvation-Induced Thermotolerance of Escherichia coli

DnaK is essential for starvation-induced resistance to heat, oxidation, and reductive division in Escherichia coli. Studies reported here indicate that DnaK is also required for starvation-induced osmotolerance, catalase activity, and the production of the RpoS-controlled Dps (PexB) protein. Because these dnaK mutant phenotypes closely resemble those of rpoS (ς³⁸) mutants, the relationship between DnaK and RpoS was evaluated directly during growth and starvation at 30°C in strains with genetically altered DnaK content. A starvation-specific effect of DnaK on RpoS abundance was observed. During carbon starvation, DnaK deficiency reduced RpoS levels threefold, while DnaK excess increased RpoS levels nearly twofold. Complementation of the dnaK mutation restored starvation-induced RpoS levels to normal. RpoS deficiency had no effect on the cellular concentration of DnaK, revealing an epistatic relationship between DnaK and RpoS. Protein half-life studies conducted at the onset of starvation indicate that DnaK deficiency significantly destabilized RpoS. RpoH (ς³²) suppressors of the dnaK mutant with restored levels of RpoS and dnaK rpoS double mutants were used to show that DnaK plays both an independent and an RpoS-dependent role in starvation-induced thermotolerance. The results suggest that DnaK coordinates sigma factor levels in glucose-starved E. coli.     

In vivo analysis of the overlapping functions of DnaK and trigger factor

Trigger factor (TF) is a ribosome-bound protein that combines catalysis of peptidyl-prolyl isomerization and chaperone-like activities in Escherichia coli. TF was shown to cooperate with the DnaK (Hsp70) chaperone machinery in the folding of newly synthesized proteins, and the double deletion of the corresponding genes (tig and dnaK) exhibited synthetic lethality. We used a detailed genetic approach to characterize various aspects of this functional cooperation in vivo. Surprisingly, we showed that under specific growth conditions, one can delete both dnaK and tig, indicating that bacterial survival can be maintained in the absence of these two major cytosolic chaperones. The strain lacking both DnaK and TF exhibits a very narrow temperature range of growth and a high level of aggregated proteins when compared to either of the single mutants. We found that, in the absence of DnaK, both the N-terminal ribosome-binding domain and the C-terminal domain of unknown function are essential for TF chaperone activity. In contrast, the central PPIase domain is dispensable. Taken together, our data indicate that under certain conditions, folding of newly synthesized proteins in E. coli is not totally dependent on an interaction with either TF and/or DnaK, and suggest that additional chaperones may be involved in this essential process.     

Expression of dnaK and groESL operons during sporulation of Bacillus megaterium

Expression of the dnaK and groEL genes during sporulation was assayed by determination of their mRNA levels by Northern blotting and compared with the relative level and rate of synthesis of the corresponding proteins. The ability of sporulating cells to respond to a heat shock by an increase in dnaK and groEL expression was determined at the same time. Synthesis of DnaK and GroEL encoding mRNAs during sporulation in non-shocked cells was low suggesting that this kind of cytodifferentiation was not accompanied by enhanced synthesis of these chaperones. Also the ability of sporulating cells to respond to a heat shock by stimulating their synthesis substantially decreased during the reversible and dropped to negligible values during the irreversible sporulation phase. Nevertheless, some dependence of the heat shock response on sporulation exists because sporulation suppression by mutation or by netropsin treatment further decreased the cells' capacity to respond to a heat shock.     

In vitro roles of Escbericbia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids

Summary Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells. Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins. Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive. Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein. This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns. These results suggest that at least one of the heat shock proteins, DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity. However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions.     

Involvement of DnaK3, One of the Three DnaK Proteins of Cyanobacterium Synechococcus sp. PCC7942, in Translational Process on the Surface of the Thylakoid Membrane

The Synechococcus sp. PCC7942 strain carrying a missense mutation in the peptide-binding domain of DnaK3, one of the essential dnaK gene products, revealed temperature-sensitive growth. We also isolated suppressor mutants of this strain. One of the suppressors was mapped in the ribosomal protein gene rpl24 (syc1876), which encodes the 50S ribosomal protein L24. Subcellular localization of three DnaK proteins was determined, and the results indicated that a quantity of DnaK3 was dislocated from membrane-bound polysomes when dnaK3 temperature-sensitive mutant was incubated at non-permissive temperatures. Furthermore, we examined the photosystem II reaction center protein D1 and detected a translational intermediate polypeptide in membrane-bound polysome fractions prepared from dnaK3 temperature-sensitive cells grown at high temperature. These characteristic features of DnaK3 localizations and detection of D1 protein intermediate were not observed in the suppressor mutant even at high temperatures.     

Heat Resistance and Salt Hypersensitivity in Lactococcus lactis Due to Spontaneous Mutation of llmg_1816 (gdpP) Induced by High-Temperature Growth

During construction of several gene deletion mutants in Lactococcus lactis MG1363 which involved a high-temperature (37.5°C) incubation step, additional spontaneous mutations were observed which resulted in stable heat resistance and in some cases salt-hypersensitive phenotypes. Whole-genome sequencing of one strain which was both heat resistant and salt hypersensitive, followed by PCR and sequencing of four other mutants which shared these phenotypes, revealed independent mutations in llmg_1816 in all cases. This gene encodes a membrane-bound stress signaling protein of the GdpP family, members of which exhibit cyclic dimeric AMP (c-di-AMP)-specific phosphodiesterase activity. Mutations were predicted to lead to single amino acid substitutions or protein truncations. An independent llmg_1816 mutant (Δ1816), created using a suicide vector, also displayed heat resistance and salt hypersensitivity phenotypes which could be restored to wild-type levels following plasmid excision. L. lactis Δ1816 also displayed improved growth in response to sublethal concentrations of penicillin G. High-temperature incubation of a wild-type industrial L. lactis strain also resulted in spontaneous mutation of llmg_1816 and heat-resistant and salt-hypersensitive phenotypes, suggesting that this is not a strain-specific phenomenon and that it is independent of a plasmid integration event. Acidification of milk by the llmg_1816-altered strain was inhibited by lower salt concentrations than the parent strain. This study demonstrates that spontaneous mutations can occur during high-temperature growth of L. lactis and that inactivation of llmg_1816 leads to temperature resistance and salt hypersensitivity.