Bacteria, as all kind of organisms, respond to a sudden increase of the temperature by a heat shock response which induces a specific set of proteins. This stress system belongs to the best-studied cellular responses. As the protein composition of a cell determines substantially the cellular functions, the heat shock response is mainly understood in terms of protein synthesis and composition.

The heat shock response of E. coli is mediated by the alternative sigma factor σ³² (rpoH gene product) and leads to induction of more than 20 heat shock proteins 12. In difference to other stress responses of E. coli which in a regulatory cascade first lead to synthesis of the respective sigma factors which then, consecutively, direct the transcription of their genes, the transcriptional induction of the heat shock proteins can start very fast after a heat shock. This first fast synthesis of heat shock proteins is transient, followed by an adaptation period with a lower rate of heat shock protein synthesis and later reaches a new steady-state level 13. This is because a cell also under non-stress conditions contains a set of a low number of σ³² molecules (about 10–30 molecules; 45). Those molecules although possessing a high binding affinity for the RNA polymerase 6, however, are rarely transcriptionally active, because of sequestering by binding to DnaK, which together with its cochaperones DnaJ and GrpE associates with σ³² 78 and mediates its degradation by the FtsH protease 9. The release of σ³² from the DnaK chaperone is regulated by the amount of missfolded proteins which may accumulate either when existing proteins lose their structure, or when new-synthesised proteins do not fold properly, which is the characteristic intracellular signal of a heat shock 10 but also may occur under strong overexpression of recombinant proteins 11. Once released from DnaK, σ³² binds to RNA polymerase, is stabilised and initiates the synthesis of the heat shock proteins. This mechanism due to the high affinity of σ³² for binding to RNA polymerase results in the synthesis of heat shock proteins to a concentration of about 5 % of the total cellular protein 1.

The physiological response of E. coli towards a heat shock is more complex than just the synthesis of heat shock proteins, including transient growth stop, changes in the cell membranes due to changes in the ratio of lipids and integral membrane proteins 12, and probably also transient DNA relaxation 13. In higher eukaryotes, one of the most immediate effects of a heat shock is an extensive disruption of the cytoskeleton 1415. From the metabolic side already early studies have shown that the maintenance energy requirement increases with increasing growth temperature 16171819 and the yield coefficient Y_X/S was observed to decrease, possibly in connection to the higher protein turnover 20. A number of authors also reported increased respiration rates in relation to the specific growth rate at higher temperatures, e.g. for A. aerogenes (Klebsiella pneumoniae) 21. (referred by 22)

The group of more than 30 heat shock proteins involves mainly proteases and chaperones, being involved in the degradation of improper folded proteins. The function of these proteins is connected to the hydrolysis of ATP, such as proteolysis by Lon, FtsH and Clp in E. coli 232425, and the action of the chaperone complexes GroEL/ES and DnaK/J/GrpE 2627. Although a number of in vitro studies have been performed revealing this ATP dependence (e.g. 28), only limited information is available about the response of the in vivo ATP level after a temperature shift. Different authors (e.g. 293031) found in higher eukaryotes a fast decrease of the ATP level to less than 50 % after a temperature up-shift parallel to induction of heat shock proteins.

In E. coli the ATP concentration has been mainly followed in steady state conditions. For instance it was reported to increase several fold in correlation with the specific growth rate 3233. In response to stress ATP or the total adenylate concentration have been followed after cold shock, where both decrease 34, after UV radiation, where the ATP level transiently increased twofold 35, with a following decrease in recA⁺ cells, which the authors relate to the action of substrate level phosphorylation. Also the levels of ATP and other nucleotides have been followed after induction of recombinant proteins by IPTG 36.

Studies with an appropriate short-time resolution of the ATP response in heat-shocked E. coli cells have not been published, although those data might be valuable to better understand the in vivo function of heat shock induced chaperones and proteases.

In response to a heat shock E. coli strongly induces the production of a number of proteins with protecting and degrading functions, which are ATP dependent. However, most of the data on protease and chaperone function were obtained by in vitro studies, and there are still nearly no data available indicating, that there is a sufficient level of ATP available after a heat shock. Rather confusing results from eukaryotes suggest, that the ATP level and the energy charge after heat shock quickly decrease, which could be seen as contradictory to the optimum conditions for the function of these proteins. Therefore it was the aim of this study to measure the response of the pool of adenosine nucleotides following a temperature up-shift in E. coli.

Aside from the measurement of the adenylate phosphorylation status we wanted to discuss the results in relation to the cellular metabolism and respiratory activity. Therefore the experiments were performed in a bioreactor, which allowed the measurement of oxygen consumption and carbon dioxide formation, as well as a fast sampling of nucleotides and medium samples.

All cultivations were performed with the E. coli K-12 strain W3110, which was a major strain in the evaluation of the E. coli stress responses. As indicated in the materials and methods section the cultivations were carried out on mineral salt medium with glucose as carbon source to avoid complex reactions in view of amino acid availability. The medium used was originally designed for high cell density fermentations, and therefore insures, that all salts are maintained at appropriate concentrations up to the end of the experiment.

The experiments were performed with a temperature shift from 30°C to 42°C. In this range the viability of the cell is not significantly affected, although the heat shock response is strongly induced. The basic growth behaviour and parameters are shown in Fig. 1 and Table 1, respectively. Cultivations started with a batch phase at 30°C for about 11 hours, corresponding to 6 generations, to ensure a steady state of cellular parameters. The specific growth rate μ was determined to be 0.35 h^-1. By following the glucose and acetate concentrations (see Fig. 1) the specific rate for glucose uptake (q_S) was determined to be 5.1 mmol g^-1 h^-1 and the specific acetate production rate (q_A) was 2.5 mmol g^-1 h^-1. The temperature up-shift to 42°C was performed at a cell density of OD₅₀₀ = 3 (corresponding to a dry cell weight of 0.675 g L^-1) within 3 min. After the temperature shift the cultivation was continued for one hour. Rapidly after the temperature shift the cells start to increase the specific growth rate to 0.70 h^-1 and correspondingly the rates for glucose uptake and acetate production (see Table 1).

During growth at 30°C the ATP concentration was detected to be about 1.5 μmol per OD₅₀₀. Concentrations of the other lower phosphorylated adenosine nucleotides were very low during this phase and the resulting adenylate energy charge (EC) was above 0.95. Nucleotide samples were collected in five minute intervals. The ATP concentration increased to 2.75 μmol per OD₅₀₀ immediately after the temperature shift to 42°C and also the ADP concentration was found to transiently increase significantly (see Fig. 2). After the transient increase the cellular concentrations of both nucleotides decreased and finally we detected a slight increase of AMP towards the end of the cultivation. Interestingly, the elevation of the ATP level was connected to a total increase of all adenosine phosphates, resulting in a slightly lowered EC to 0.8 (see Fig. 3). However, similar to ATP, also the sum of adenosine phosphates increased only transiently, approaching to the pre-shift level about 20 min after the temperature up-shift. The slight increase of AMP to the end of the cultivation influenced the energy charge, which decreased to 0.65. The transient increase of ATP after the temperature up-shift is connected to a high respiratory activity (Fig. 1C) and occurs in the phase, where also the flux of glucose is rapidly increasing.

In alternate series of experiments the temperature was shifted either from 37°C to 48°C, or from 35°C to 55°C which leads to growth inhibition and cell death. In both cases the temperature up-shift caused a strong transient increase of respiration and, at least in the first case we also found the rapid increase of ATP, however, the following reduction of the ATP level was much faster (data not shown).

Further it was investigated how the time kinetics of the adenosine nucleotides are related to the expression profiles of typical heat shock genes. These experiments were performed in shake flasks with on-line monitoring of dissolved oxygen and temperature. A fast temperature up-shift was reached by diluting the culture with pre-heated medium which explains the decrease of OD₅₈₀ in Fig. 4. Interestingly, the induction of the three investigated heat shock controlled genes was different. The expression of dnaK and ibp were both induced very rapidly and reached their maximum mRNA levels within only 1 min after the heat shock, but lon reached the maximum RNA level after 5 min. A quantitative evaluation of the mRNA amounts showed the highest concentration for ibp, but lower expression of dnaK and the lowest for lon. The level of dnaK mRNA was continuously significantly higher expressed than before the shift, but the mRNAs of ibp and lon were very low or not any more detectable after only 10 min. Interestingly, this shut-off of the expression of these genes is at about the same time when the pre-shift level of nucleotides was re-established.

Our studies indicate, that E. coli responds to a temperature up-shift by rapidly increasing respiration and glucose uptake. The increase of glucose uptake is possibly mainly directed to glycolysis and, consequently, to energy production. Concomitantly we detected a significant drop in the pH and an increase of the acetate level. The higher production of acetate per glucose (Y_A/S) might point to an increase of glycolytic activity, and suggests that the flow to the tricarboxylic acid cycle was not increased correspondingly.

We detected a significant increase of the concentration of the sum of adenosine nucleotides (AXP) directly during the temperature up-shift which possibly is caused by RNA degradation as is concluded from the total RNA concentrations per cell which decreased to less than 50 % if the samples before and after the temperature up-shift are compared (data not shown). This increase of the AXP concentration was connected to a decrease in the energy charge, as the increase of ADP was higher than the increase of ATP. It remains to be investigated whether this relative transient increase of ADP has physiological effects. For example it might be hypothesized that the high concentration of ADP transiently inactivates various chaperones (see e.g.3738) which bind ADP with higher affinity compared to ATP.

After the heat shock the cells strongly increase respiration and CO₂ production which should be related to ain increased glycolytic flux, as correspondingly acetate formation is increased. A similar behaviour of increased glycolytic flux we have observed from other cultivations where a stress was caused by strong induction of a recombinant protein and also provoked a transient increase of the ATP level and higher respiration. However, differently from the investigated temperature up-shift, after induction of the recombinant protein the cells lost their viability 39. Also a lethal heat shock caused by a temperature up-shift from 37°C to 52°C 40 or from 37°C to 48°C (own results, not shown) resulted in a transient increase of ATP, but this was much slower and the increase was only about 30 % with a maximum at about 30 min after the temperature shift.

Our data suggest that similarly as known for the heat shock response, that the response of the nucleotides is transient and the energy charge is restored 15 to 20 min after the heat shock. After this transient induction of the heat shock response cell growth recovers and it may be hypothesised that the cellular synthetic activities and metabolite pools reach a new steady state.

This study shows that the heat shock response in E. coli in connection to the adenosine phosphates is different from the response of eukaryotes. Whereas different authors described in eukaryotes an immediate decrease of the ATP level following a temperature up-shift (e.g. 293031), in E. coli we found a transient increase, before the ATP level is lowered. It may be hypothesised, but has to be verified by further experiments that this transient increase of the ATP concentration is important for the action of chaperones and proteases and consequently for the function of the cellular system towards a heat shock response.

SW and PN carried out the fermentations and nucleotide analyses. JS, CF, CM and DB developed the method for analysing the mRNAs and performed the corresponding analysis. JP and AV were included in the wireless analysis with the Senbit system. JS, PN and AV drafted the manuscript. All authors read and approved the final manuscript.