Cross-Talk between the Canonical and the Nitrogen-Related Phosphotransferase Systems Modulates Synthesis of the KdpFABC Potassium Transporter in <b><i>Escherichia coli</i></b>

Abstract

Many Proteobacteria possess the regulatory nitrogen-related phosphotransferase system (PTS^Ntr), which operates in parallel to the transport PTS. PTS^Ntr is composed of the proteins EI^Ntr and NPr and the final phosphate acceptor EIIA^Ntr. Both PTSs can exchange phosphoryl groups among each other. Proteins governing K⁺ uptake represent a major target of PTS^Ntr in Escherichia coli. Nonphosphorylated EIIA^Ntr binds and stimulates the K⁺ sensor KdpD, which activates expression of the kdpFABC operon encoding a K⁺ transporter. Here we show that this regulation also operates in an ilvG⁺ strain ruling out previous concern about interference with a nonfunctional ilvG allele present in many strains. Furthermore, we analyzed phosphorylation of EIIA^Ntr. In wild-type cells EIIA^Ntr is predominantly phosphorylated, regardless of the growth stage and the utilized carbon source. However, cross-phosphorylation of EIIA^Ntr by the transport PTS becomes apparent in the absence of EI^Ntr: EIIA^Ntr is predominantly nonphosphorylated when cells grow on a PTS sugar and phosphorylated when a non-PTS carbohydrate is utilized. These differences in phosphorylation are transduced into corresponding kdpFABC transcription levels. Thus, the transport PTS may affect phosphorylation of EIIA^Ntr and accordingly modulate processes controlled by EIIA^Ntr. Our data suggest that this cross-talk becomes most relevant under conditions that would inhibit activity of EI^Ntr.

Non‐canonical activation of histidine kinase KdpD by phosphotransferase protein PtsN through interaction with the transmitter domain

“…S10A). Indeed, an approximately sevenfold lower activity was observed in case of the PtsN‐H73E variant as compared to PtsN‐H73A and this activity was even slightly lower as observed for wild‐type PtsN, which was previously shown to predominantly prevail in its phosphorylated form under standard growth conditions (Bahr et al ., ; Lüttmann et al ., ). In addition, we used SPR spectroscopy to test binding of purified Strep‐PtsN‐H73E to the cytoplasmic C‐terminal portion of KdpD (Fig.…”

“…Notably, only non‐phosphorylated PtsN is able to efficiently bind and regulate TrkA and KdpD in E. coli (Lee et al ., ; Lüttmann et al ., ). Recent results indicate that the phosphorylation state of PTS Ntr is controlled by nitrogen and carbohydrate availability suggesting that PTS Ntr coordinates K + uptake with the metabolic state of the cell (Pflüger & de Lorenzo, ; Lee et al ., ; Lüttmann et al ., ; Ronneau et al ., ). Regulation of KdpD by interaction with PtsN was also observed in Pseudomonas putida and Rhizobium leguminosarum emphasizing its importance in a wide range of species (Prell et al ., ; Deuschle et al ., ).…”

“…A) resulting in activation of KdpD/KdpE irrespective of the K + signal (Heermann et al ., ). Moreover, the phosphotransferase protein PtsN (alternative name EIIA Ntr ) activates the KdpD/KdpE TCS by binding to KdpD, boosting kdpFABC expression (Lüttmann et al ., ). PtsN is member of the PTS Ntr , which is an abridged PEP‐dependent phosphotransferase system (PTS) that is conserved in Proteobacteria and operates in parallel to the canonical PTS dedicated to carbohydrate uptake (Pflüger‐Grau & Görke, ).…”

“…Consequently, ptsN mutants are disturbed in K + homeostasis (Lee et al ., ). Notably, only non‐phosphorylated PtsN is able to efficiently bind and regulate TrkA and KdpD in E. coli (Lee et al ., ; Lüttmann et al ., ). Recent results indicate that the phosphorylation state of PTS Ntr is controlled by nitrogen and carbohydrate availability suggesting that PTS Ntr coordinates K + uptake with the metabolic state of the cell (Pflüger & de Lorenzo, ; Lee et al ., ; Lüttmann et al ., ; Ronneau et al ., ).…”

Crystallization and preliminary X-ray diffraction analysis of YhbJ fromEscherichia coli, a key protein involved in the GlmYZ sRNA regulatory cascade

“…This plasmid is a pBR322 derivative and carries the recombinant strep-yhbJ gene under control of the IPTGinducible P tac promoter, the lacI q gene for control of the P tac promoter and the bla ampicillin-resistance gene (Lü ttmann et al, 2012). Strep-tagged YhbJ was overproduced in E. coli strain Z106 that is deficient in the genes of glmY and glmZ.…”