Escherichia coli enzyme IIA ^Ntr regulates the K ⁺ transporter TrkA

Abstract

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

Salmonella pathogenicity island 2 expression negatively controlled by EIIA Ntr –SsrB interaction is required for Salmonella virulence

“…The interactions between EIIA Ntr and its partner proteins are determined by the phosphorylation state of EIIA Ntr . For interaction with the TrkA or KdpD protein, EIIA Ntr should be dephosphorylated (18,19). We found that Salmonella EIIA Ntr carrying a mutation in the putative phosphorylation site (i.e., H73A substitution) can still interact with SsrB in vivo and maintain the normal levels of SPI-2 gene expression (Fig.…”

“…The ptsN gene encodes EIIA Ntr , a component of the nitrogen-metabolic PTS (20). Because it has been reported that EIIA Ntr performs gene regulatory function in E. coli (18,19) and Pseudomonas putida (21), we wanted to search for Salmonella genes whose expression levels are affected by EIIA Ntr . We conducted DNA microarray analysis using RNA isolated from wild-type and ptsN deletion strains grown in Luria-Bertani broth (LB) medium to stationary phase.…”

“…EIIA Ntr has been reported to bind directly to the TrkA and KdpD proteins and to control K + homeostasis in E. coli (18,19). Therefore, we investigated whether Salmonella EIIA Ntr interacts with the SsrB protein using a bacterial two-hybrid system (24).…”

“…In Escherichia coli, the ptsN gene encoding EIIA Ntr was identified as a suppressor of some lethal phenotypes; inactivation of the ptsN gene recovered viability of an era mutant lacking essential GTPase activity (16), whereas overexpression of EIIA Ntr compensated for the loss of σ E activity (17). In addition, E. coli EIIA Ntr was reported to interact directly with the TrkA potassium (K + ) transporter and the KdpD sensor kinase to control cytoplasmic K + levels (18,19).…”

Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids

“…Pseudoauxotrophy is known in E. coli, where provision of leu-containing peptides reduces expression of the first step in LIV biosynthesis (acetohydroxy acid synthase) (17), and bacteria require addition of ile and val. Legumes might impose a similar selection on bacteroids by provision of peptides and gain control over their development and persistence by regulating the supply of LIV.…”