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Burg bei Magdeburg, Germany

Sharma P.,University of Amsterdam | Stagge S.,MPI fur Dynamik Komplexer Technischer Systeme | Bekker M.,University of Amsterdam | Bekker M.,The Coca-Cola Company | And 2 more authors.

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in 'respiratory' electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form. © 2013 Sharma et al. Source

Steinsiek S.,MPI fur Dynamik Komplexer Technischer Systeme | Bettenbrock K.,MPI fur Dynamik Komplexer Technischer Systeme
Journal of Bacteriology

In Escherichia coli, several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but the mannose PTS and the galactose and maltose transporters also can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been investigated previously because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit have not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as glucose-limited continuous cultivations. Deletion of EIIBC disturbs glucose transport severely in batch cultures; cyclic AMP (cAMP)-cAMP receptor protein (CRP) levels rise, and induction of the mgl operon occurs. Nevertheless, Mgl activity is not essential for growth of these mutants, since deletion of this transporter did not affect the growth rate; the activities of the remaining transporters seem to be sufficient. Under conditions of glucose limitation, mgl is upregulated 23-fold compared to levels for growth under glucose excess. Despite the strong induction of mgl upon glucose limitation, deletion of this transport system did not lead to further changes. Although the galactose transporters are often regarded as important for glucose uptake at micromolar concentrations, the glucose as well as mannose PTS might be sufficient for growth at this relatively low dilution rate. © 2012, American Society for Microbiology. Source

Genzel Y.,MPI fur Dynamik Komplexer Technischer Systeme | Frensing T.,MPI fur Dynamik Komplexer Technischer Systeme | Reichl U.,MPI fur Dynamik Komplexer Technischer Systeme
Chemie in Unserer Zeit

Today, about 95 % of the available influenza vaccine doses are still produced in embryonated chicken eggs. However, since the 2009 H1N1 influenza pandemic many people have realized that in case of a real threat, it might be difficult to guarantee the timely supply of a sufficient amount of vaccines. Therefore, it is of utmost importance to improve existing processes and to develop alternative manufacturing methods. The cell culture-based processes discussed in this manuscript represent a promising option to establish scalable and robust production processes. Probably these processes have not been pushed up to their limits yet. Further optimization concerning the design of bioprocesses and the optimization of biological systems involved could contribute significantly to increase yields and product quality. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Steinsiek S.,MPI fur Dynamik Komplexer Technischer Systeme | Frixel S.,MPI fur Dynamik Komplexer Technischer Systeme | Stagge S.,MPI fur Dynamik Komplexer Technischer Systeme | Bettenbrock K.,MPI fur Dynamik Komplexer Technischer Systeme
Journal of Biotechnology

Depending on the availability of oxygen, Escherichia coli is able to switch between aerobic respiratory metabolism and anaerobic mixed acid fermentation. An important, yet understudied, metabolic mode is the micro-aerobic metabolism at intermediate oxygen availabilities. The relationship between oxygen input, physiology and gene expression of E. coli MG1655 and two isogenic mutants lacking succinate dehydrogenase (SDH) and fumarate reductase (FRD) activities was analyzed at different aerobiosis levels. Growth rate and cell yield were very similar to the parent strain. By-product formation was altered in the sdhC mutant to higher acetic acid and glutamate production in batch cultures. In continuous cultures with defined oxygen input gene expression analysis revealed a dependency of many catabolic genes to aerobiosis. Acetate excretion was still detectable under aerobic conditions in the sdhC mutant; the frdA mutant lacked anaerobic succinate excretion. Anaerobic repression of the sdh operon was diminished in the frdA strain, possibly to allow SDH to partially replace FRD. The experiments illustrate the remarkable adaptability of E. coli physiology-to compensate for the absence of important metabolic genes by altering carbon flux and/or gene expression such that there are only minor changes in growth capability across the aerobiosis range. © 2011 Elsevier B.V. Source

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