The maintenance of ionic homeostasis in response to changes in the environment is essential for all living cells. Although there are still many important questions concerning the role of the major monovalent cation K ؉ , cytoplasmic K ؉ in bacteria is required for diverse processes. Here, we show that enzyme IIA Ntr (EIIA Ntr ) of the nitrogen-metabolic phosphotransferase system interacts with and regulates the Escherichia coli K ؉ transporter TrkA. Previously we reported that an E. coli K-12 mutant in the ptsN gene encoding EIIA Ntr was extremely sensitive to growth inhibition by leucine or leucine-containing peptides (LCPs). This sensitivity was due to the requirement of the dephosphorylated form of EIIA Ntr for the derepression of ilvBN expression. Whereas the ptsN mutant is extremely sensitive to LCPs, a ptsN trkA double mutant is as resistant as WT. Furthermore, the sensitivity of the ptsN mutant to LCPs decreases as the K ؉ level in culture media is lowered. We demonstrate that dephosphorylated EIIA Ntr , but not its phosphorylated form, forms a tight complex with TrkA that inhibits the accumulation of high intracellular concentrations of K ؉ . High cellular K ؉ levels in a ptsN mutant promote the sensitivity of E. coli K-12 to leucine or LCPs by inhibiting both the expression of ilvBN and the activity of its gene products. Here, we delineate the similarity of regulatory mechanisms for the paralogous carbon and nitrogen phosphotransferase systems. Dephosphorylated EIIA Glc regulates a variety of transport systems for carbon sources, whereas dephosphorylated EIIA Ntr regulates the transport system for K ؉ , which has global effects related to nitrogen metabolism. leucine toxicity ͉ nitrogen-metabolic phosphotransferase system (PTS) ͉ potassium transporter TrkA ͉ protein-protein interaction ͉ signal transduction T he well defined phosphotransferase system (PTS) is composed of two general cytoplasmic proteins, enzyme I (EI) and histidine phosphocarrier protein (HPr), and some sugarspecific components collectively known as enzymes II (1). The carbohydrate PTS, especially for glucose uptake, occupies a central position in bacterial physiology as a result of the identification of multiple regulatory functions superimposed on the transport functions, such as regulation of chemotaxis by EI (2); regulation of glycogen breakdown by HPr (3, 4); regulation of the global repressor Mlc by the membrane-bound glucose transporter EIICB Glc (5-7); and regulation of carbohydrate transport and metabolism (1,8,9), the metabolic flux between fermentation and respiration (10), and adenylyl cyclase activity (11) by EIIA Glc . These regulatory functions of the carbohydrate PTS depend on the phosphorylation state of the involved components, which have been shown to increase in the absence and decrease in the presence of a PTS sugar substrate.The nitrogen-metabolic PTS consists of enzyme I Ntr (EI Ntr , an
contributed equally to this workIn addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes. Exposure of cells to glucose can result in repression or induction of gene expression. While the mechanism for carbon catabolite repression by glucose was well documented, that for glucose induction was not clearly understood in Escherichia coli. Recently, glucose induction of several E.coli genes has been shown to be mediated by the global repressor Mlc. Here, we elucidate a general mechanism for glucose induction of gene expression in E.coli, revealing a novel type of regulatory circuit for gene expression mediated by the phosphorylation state-dependent interaction of a membrane-bound protein with a repressor. The dephospho-form of enzyme IICB Glc , but not its phospho-form, interacts directly with Mlc and induces transcription of Mlc-regulated genes by displacing Mlc from its target sequences. Therefore, the glucose induction of Mlc-regulated genes is caused by dephosphorylation of the membrane-bound transporter enzyme IICB Glc , which directly recruits Mlc to derepress its regulon.
SummaryWhile the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogenmetabolic phosphotransferase system (nitrogenmetabolic PTS). To elucidate the physiological role of the nitrogen-metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K-12 strain MG1655 deleted for the ptsP gene encoding the first enzyme of the nitrogen-metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIA Ntr ) mutant became extremely sensitive to leucine-containing peptides (LCPs), while both ptsP (encoding enzyme I Ntr ) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity of LCPs was found to be due to leucine and the dephospho-form of enzyme IIA Ntr was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho-form of enzyme IIA Ntr is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched-chain amino acids.
We have developed the first selective fluorescent chemosensor (PyDPA) for (p)ppGpp, a bacterial and plant alarmone. By using pyrene-excimer fluorescence, PyDPA shows very good selectivity for (p)ppGpp from among other nucleotides in water. PyDPA was used for the real-time detection of in vitro ppGpp synthesis by bacterial ribosomal complexes.
SummaryAn Escherichia coli mutant devoid of enzyme IIA Ntr (EIIA Ntr ) of the nitrogen PTS is extremely sensitive to leucine-containing peptides due to decreased expression of acetohydroxy acid synthase. This decreased expression is due to defective potassium homeostasis. We further elucidate here the mechanism for regulation of gene expression by the intracellular level of K + . The leucine hypersensitivity of a ptsN (encoding EIIA Ntr ) mutant was suppressed by deleting rpoS, encoding the stationary phase s factor. Despite intracellular levels of sigma factors comparable to the wild-type strain, most of the genes downregulated in a ptsN mutant are controlled by s 70 , while all the upregulated genes are controlled by s S , implying that the balance of sigma activities is modified by ptsN deletion. This change of sigma factor activities in the deletion mutant was found to be due to increased levels of K + . In vitro transcription assays demonstrated that a s 70 controlled gene and a s S controlled gene were differentially affected by potassium concentration. Biochemical studies revealed that K + is responsible for sigma factor competition by differentially influencing the binding of s 70 and s S to core RNA polymerase. Taken together, the data suggest that EIIA Ntr controls sigma factor selectivity by regulating the intracellular K + level.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.