Project description:Up-regulation of motility genes and biosynthesis genes were found in the pck expressing high ATP cell by transcriptome analysis while catabolism genes were down-regulated. Keywords: response depends on intracellular ATP concentration derived by pck or ppc overexpression 1. pck or ppc overexpressing E. coli at early log phase using glucose-minimal medium 2. pck or ppc overexpressing E. coli at chemostat culture (D=0.1 h-1)using LB-glucose medium
Project description:Up-regulation of motility genes and biosynthesis genes were found in the pck expressing high ATP cell by transcriptome analysis while catabolism genes were down-regulated. Keywords: response depends on intracellular ATP concentration derived by pck or ppc overexpression
Project description:To investigate the extent of the effect of poly(A) polymerase (PAP I)-mediated polyadenylation on RNA stability, we performed the first genome-wide study of RNA stability in the absence of PAP I activity. Inactivation of the pcnB gene coding for PAP I led to a global stabilization of E. coli RNAs, with 1403 stabilized transcripts and only 4 destabilized. Stabilized RNAs were involved in essential cellular functions such as DNA replication and repair, translation, RNA degradation, central carbon metabolism but also in stress responses. Because PAP I is an ATP-consuming enzyme we wondered whether the RNA stabilization observed after inactivation of PAP I could also be related to changes in intracellular ATP levels. We demonstrated for the first time in E. coli, that lowering intracellular ATP levels below 1 µM/OD stabilizes RNAs. Although the ATP level was reduced by 20 % in the pcnB mutant on glucose, the ATP level was still too high to have any role in the observed RNA stabilization. However, in experiments where the ATP level was artificially strongly decreased, inactivation of PAP I by substrate availability was implicated in the stabilization mechanism of certain RNAs. This study clearly demonstrates that PAP I is at the crossroads of the regulation of RNA stability by energy status in E. coli cells.
Project description:We have previously reported that phosphoenolpyruvate carboxykinase(Pck) overexpression under glycolytic conditions enables Escherichia coli to harbor a high intracellular ATP pool resulting in enhanced recombinant protein synthesis and biohydrogen production. To understand possible reasons of the high ATP haboring cell, we carried out transcriptome and metabolic flux analysis.
Project description:PurA encodes an adenylosuccinate synthetase that catalyzes the first step in the de novo synthesis of AMP. A deficiency in purA has been found to confer Escherichia coli tolerance to diverse types of antibiotics. To understand the molecular basis of the increased tolerance, we performed RNA-seq analysis to compare the transcriptional profiles of wild-type and ΔpurA mutant strains before and after ciprofloxacin treatment. The data showed that the purA deficiency suppresses transcript levels from NADH:quinone oxidoreductase genes prior to stress exposure, thereby predisposing E. coli to antibiotic tolerance by reducing respiration. During ciprofloxacin exposure, a purA deficiency suppressed a surge in expression of TCA cycle and ATP synthesis genes and the accumulation of intracellular ATP and ROS, thereby conferring bacterial tolerance to diverse antibiotic stresses.
Project description:The post-transcriptional process of RNA polyadenylation sits at the crossroads of energy metabolism and RNA metabolism. RNA polyadenylation is catalyzed by poly(A) polymerases which use ATP as a substrate to add adenine residues to the 3’ end of RNAs, which can alter their stability. In E. coli, RNA polyadenylation mediated by the major poly(A) polymerase (PAP I) was previously shown to facilitate degradation of individual RNAs. In this study, we performed the first ever genome-wide study of RNA stability in the absence of PAP I. Inactivation of the pcnB gene coding for PAP I led to the stabilization of more than a thousand of E. coli RNAs in the form of full-length functional molecules or non-functional fragments, involved in essential cellular functions such as DNA replication and repair, translation, central carbon metabolism, and stress response. The absence of PAP I also altered the energy metabolism, with an almost 20 % reduction in ATP levels. To better understand how RNA and energy metabolisms are interconnected, we investigated the role of ATP levels in regulating RNA stability. When we lowered intracellular ATP levels below 0.5 mM, certain RNAs were stabilized demonstrating the causal link between ATP levels and RNA stability for the first time in E. coli. Above this concentration, changes in ATP levels had no impact on RNA stability. We also demonstrated that some RNAs were stabilized when PAP I was inactivated by low ATP availability. These results clearly demonstrate that PAP I mediates an energy-dependent RNA stabilization which may contribute to cell energy homeostasis under energy-limited conditions.