Stressantwort von Mikroorganismen (Volume 55) (English shop)

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The investigation of the cellular stress response is carried out on several levels, including the genome, proteome and metabolome levels. The metabolome level provides insight into the processes within the cells, namely into the way in which the metabolic fluxes in the cell are redirected in order to respond to an external factor. Knowledge of this cellular response helps to understand larger interrelationships within the cells and to apply them for a targeted modification of microorganisms, e.g. for improved protein production.

The investigation of the intracellular metabolites in the cells at a defined time point of the cultivation requires a rapid arrest of the cellular processes. For this purpose, cold solutions, so-called quenching solutions, mostly based on the organic solvent methanol, are generally used. In all cases investigated so far, the quenching process leads to the loss of intracellular metabolites such as ATP. The effect of the solutions on the integrity of the cells was investigated in the present work. Escherichia coli and Saccharomyces cerevisiae were used as organisms. Both the effects of different starting temperatures of the quenching solution and the effect of different compositions were investigated. For this purpose, flow cytometry for the determination of the damaged cell population fractions, the comparison of the optical density before and after the treatment (indicator of cell lysis), as well as the determination of a representative metabolite (ATP) in the quenching solution supernatant and cell extract were established and applied.

With the quenching solution having the smallest effect on the cells, the long-term stress response of S. cerevisiae and of some selected knockout mutants (mae1Δ, zwf1Δ) was subsequently investigated. The cultivation was carried out in shake flasks, with the minimal medium being supplemented with the selected stress factors diamide (oxidative stress) or caffeine. By means of metabolic profiling, the cell extracts were analysed using GC-MS. It turned out that diamide at the concentration used had no long-term effect on the cell metabolism. Caffeine, however, strongly disturbed the cells, which was reflected in a markedly reduced cell growth, altered amino acid and lower fatty acid concentrations, as well as in the production of compatible solutes.

For the investigation of the dynamic stress response (i.e. the application of a pulse), an automated sampling system for glass reactors was developed (rapid sampling), with the aim of being used in a continuously operated bioreactor. The system allows a continuous sample flow to be divided into individual samples, each covering a time span of approx. 143 ms.