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Regulation von ABC-Transportern in Escherichia coli und Thermus thermophilus

Regulation von ABC-Transportern in Escherichia coli und Thermus thermophilus

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LENGSFELD, Christina, 2009. Regulation von ABC-Transportern in Escherichia coli und Thermus thermophilus [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Lengsfeld2009Regul-7160, title={Regulation von ABC-Transportern in Escherichia coli und Thermus thermophilus}, year={2009}, author={Lengsfeld, Christina}, address={Konstanz}, school={Universität Konstanz} }

Lengsfeld, Christina application/pdf deu Regulation von ABC-Transportern in Escherichia coli und Thermus thermophilus Glucose and Glucokinase controlled mal gene expression in Escherichia coli.<br />MalT is the central transcriptional activator of all mal genes in Escherichia coli. It can be controlled (activated) by the inducer maltotriose, it can be inhibited by the interaction with certain proteins and its expression can be controlled. Here we report a novel aspect of mal gene regulation: the effect of cytoplasmic glucose and glucokinase on the activity of MalT as well as its expression. Amylomaltase is essential for the metabolism of maltose. It forms maltodextrins and glucose from maltose or maltodextrins in such a way that the number of glucosidic linkages stays constant. We found that glucose above a concentration of 0.1 mM blocked the activity of the enzyme. It is known that malQ mutants when grown in the absence of maltose or maltodextrins are endogenously induced by maltotriose that is derived from the degradation of glycogen. Therefore, the fact that glk malQ+ mutants showed elevated mal gene expression finds its explanation in the reduced ability to remove glucose from MalQ-catalyzed maltodextrin formation and is thus caused by the formation of a metabolically induced MalQ- phenotype. However, even in mutants lacking glycogen glucokinase controls endogenous induction. We found that overexpressed glucokinase due to its structural similarity with Mlc (the repressor of malT) competes at the level of PtsG with Mlc. In addition, even in mutants lacking Mlc (and glycogen) the overexpression of glk leads to a reduction in mal gene expression. This repression was dependent on the presence of either maltodextrin phosphorylase or amylomaltase and led to the inactivation of MalT.<br />Network regulation of sugar transport in Thermus thermophilus.<br />We report the presence of Mlc in a thermophilic bacterium. Mlc is known as a global regulator of sugar metabolism in gram-negative enteric bacteria that is controlled by sequestration to a glucose-transporting EIIBGlc of the phosphotransferase system (PTS). Since thermophilic bacteria do not possess PTS, Mlc in Thermus thermophilus must be differently controlled. DNA sequence alignments between Mlc from T. thermophilus (MlcTth) and Mlc from E. coli (MlcEco) revealed that MlcTth conserved five residues of the glucose binding motif of glucokinases. Here we show that MlcTth is not a glucokinase but is indeed able to bind glucose, unlike MlcEco. We found that mlc of T. thermophilus is the first gene within an operon encoding an ABC transporter for glucose, galactose and mannose, including a glucose/galactose/mannose-binding protein and two permeases. malK1, encoding the cognate ATP-hydrolyzing subunit, is located elsewhere on the chromosome. The system transports glucose at 70°C with a Km of 0.15 µM and a Vmax of 4.22 nmol per min per ml at an optical density (OD) of 1. MlcTth negatively regulates itself and the entire glucose/galactose/mannose ABC transport system operon but not malK1, with glucose acting as an inducer. MalK1 is shared with the ABC transporter for trehalose, maltose, sucrose, and palatinose (TMSP) and the ABC transporter for maltodextrins (MDX). Mutants lacking malK1 do not transport either glucose or maltose. The TMSP transporter is also able to transport glucose with a Km of 1.4 µM and a Vmax of 7.6 nmol per min per ml at an OD of 1, but it does not transport mannose. terms-of-use Lengsfeld, Christina 2011-03-24T17:32:18Z Regulation of ABC transport systems in Escherichia coli and Thermus thermophilus 2011-03-24T17:32:18Z 2009

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