Analysis of global control of Escherichia coli carbohydrate uptake

<p>Abstract</p> <p>Background</p> <p>Global control influences the regulation of many individual subsystems by superimposed regulator proteins. A prominent example is the control of carbohydrate uptake systems by the transcription factor Crp in <it>Escherichia coli</it>. A detailed understanding of the coordination of the control of individual transporters offers possibilities to explore the potential of microorganisms e.g. in biotechnology.</p> <p>Results</p> <p>An o.d.e. based mathematical model is presented that maps a physiological parameter – the specific growth rate – to the sensor of the signal transduction unit, here a component of the bacterial phosphotransferase system (PTS), namely EIIA^<it>Crr</it>. The model describes the relation between the growth rate and the degree of phosphorylation of EIIA ^{<it>crr </it>}for a number of carbohydrates by a distinctive response curve, that differentiates between PTS transported carbohydrates and non-PTS carbohydrates. With only a small number of kinetic parameters, the model is able to describe a broad range of experimental steady-state and dynamical conditions.</p> <p>Conclusion</p> <p>The steady-state characteristic presented shows a relationship between the growth rate and the output of the sensor system PTS. The glycolytic flux that is measured by this sensor is a good indicator to represent the nutritional status of the cell.</p

Is energy excess the initial trigger of carbon overflow metabolism? Transcriptional network response of carbon-limited Escherichia coli to transient carbon excess

Background: Escherichia coli adapted to carbon-limiting conditions is generally geared for energy-efficient carbon utilization. This includes also the efficient utilization of glucose, which serves as a source for cellular building blocks as well as energy. Thus, catabolic and anabolic functions are balanced under these conditions to minimize wasteful carbon utilization. Exposure to glucose excess interferes with the fine-tuned coupling of anabolism and catabolism leading to the so-called carbon overflow metabolism noticeable through acetate formation and eventually growth inhibition. Results: Cellular adaptations towards sudden but timely limited carbon excess conditions were analyzed by exposing slow-growing cells in steady state glucose-limited continuous culture to a single glucose pulse. Concentrations of metabolites as well as time-dependent transcriptome alterations were analyzed and a transcriptional network analysis performed to determine the most relevant transcription and sigma factor combinations which govern these adaptations. Down-regulation of genes related to carbon catabolism is observed mainly at the level of substrate uptake and downstream of pyruvate and not in between in the glycolytic pathway. It is mainly accomplished through the reduced activity of CRP-cAMP and through an increased influence of phosphorylated ArcA. The initiated transcriptomic change is directed towards down-regulation of genes, which contribute to active movement, carbon uptake and catabolic carbon processing, in particular to down-regulation of genes which contribute to efficient energy generation. Long-term changes persisting after glucose depletion and consumption of acetete encompassed reduced expression of genes related to active cell movement and enhanced expression of genes related to acid resistance, in particular acid resistance system 2 (GABA shunt) which can be also considered as an inefficient bypass of the TCA cycle. Conclusions: Our analysis revealed that the major part of the trancriptomic response towards the glucose pulse is not directed towards enhanced cell proliferation but towards protection against excessive intracellular accumulation of potentially harmful concentration of metabolites including among others energy rich compounds such as ATP. Thus, resources are mainly utilized to cope with “overfeeding” and not for growth including long-lasting changes which may compromise the cells future ability to perform optimally under carbon-limiting conditions (reduced motility and ineffective substrate utilization)

SYSTEMORIENTIERTE BIOPROZESSTECHNIK: INTERDISZIPLINÄRE FORSCHUNG IN BIOLOGIE, SYSTEM- UND COMPUTERWISSENSCHAFTEN

Die aktuelle Forschung in der molekularen Genetik und die Erfolge bei der Analyse von Genexpression und Proteinfunktion führen zu einer bisher unerreichten Fülle von Informationen über biologische Phänomene. Damit ergeben sich neben der medizinischen Anwendung auch neue Möglichkeiten und Aufgaben in der biotechnologischen Produktion von Wirkstoffen. Um dieses biologische Potenzial voll ausschöpfen zu können, bedarf es jedoch verstärkt interdisziplinärer Forschung in Biologie, System- und Computerwissenschaften. Der hier skizzierte Forschungsansatz soll langfristig zum Aufbau eines „Virtuellen Biologischen Labors“ führen, in dem Experimente am Rechner analog zu Experimenten im Labor durchgeführt werden können. Damit steht in Forschung und Lehre ein Werkzeug zur Vermittlung quantitativer und qualitativer Aspekte von zellulären Stoffwechsel- und Regulationsvorgängen zur Verfügung

All three quinone species play distinct roles in ensuring optimal growth under aerobic and fermentative conditions in <i>E</i>. <i>coli</i> K12 - Fig 3

<p><b>Aerobic (A) and anaerobic (B) gene expression analysis</b>. Genes encoding the terminal oxidases cytochrome bd II (<i>appC</i>), cytochrome bd (<i>cydA</i>) and cytochrome bo₃ (<i>cyoA</i>) and dehydrogenases NADH: quinone oxidoreductase II (<i>ndh</i>), NADH: ubiquinone oxidoreductase (<i>nuoN</i>) and succinate dehydrogenase (<i>sdhD)</i> were analysed. The transcription pattern was normalized to the reference genes <i>recA</i> and <i>rpoD</i> and to the expression of the wild type strain MG1655 under aerobic (A) and anaerobic (B) conditions respectively. Due to the normalization, constant or unchanged relative gene expression levels are calculated as 1. The Y-axis was formatted in logarithmic scale with base 2, to equally visualize up and downregulation of genes.</p

All three quinone species play distinct roles in ensuring optimal growth under aerobic and fermentative conditions in <i>E</i>. <i>coli</i> K12 - Fig 2

<p><b>Quinone content of the wild type MG1655 and the mutants during exponential growth under aerobic (A) and anaerobic (B) batch conditions.</b> The values represent the average values from at least three independent experiments.</p

Relative ArcA phosphorylation and quinone distribution under aerobic (+O₂) and anaerobic (-O₂) conditions.

<p>Relative ArcA phosphorylation and quinone distribution under aerobic (+O₂) and anaerobic (-O₂) conditions.</p

Reactive oxygen species in quinone mutants.

<p>Shown are mean fluorescence values of MG1655 and the three mutant strains after aerobic incubation with CellROX Green. Samples of aerobically growing cultures of the strains were taken from exponential growth phase and incubated with CellROX Green. The values represent average values from at least three independent growth curves.</p

Primer sequences.

<p>Primer sequences.</p