Insight into Bacterial Phosphotransferase System-Mediated Signaling by Interspecies Transplantation of a Transcriptional Regulator

Abstract

The bacterial sugar:phosphotransferase system (PTS) delivers phosphoryl groups via proteins EI and HPr to the EII sugar transporters. The antitermination protein LicT controls ␤-glucoside utilization in Bacillus subtilis and belongs to a family of bacterial transcriptional regulators that are antagonistically controlled by PTS-catalyzed phosphorylations at two homologous PTS regulation domains (PRDs). LicT is inhibited by phosphorylation of PRD1, which is mediated by the ␤-glucoside transporter EII Bgl . Phosphorylation of PRD2 is catalyzed by HPr and stimulates LicT activity. Here, we report that LicT, when artificially expressed in the nonrelated bacterium Escherichia coli, is likewise phosphorylated at both PRDs, but the phosphoryl group donors differ. Surprisingly, E. coli HPr phosphorylates PRD1 rather than PRD2, while the stimulatory phosphorylation of PRD2 is carried out by the HPr homolog NPr. This demonstrates that subtle differences in the interaction surface of HPr can switch its affinities toward the PRDs. NPr transfers phosphoryl groups from EI Ntr to EIIA Ntr . Together these proteins form the paralogous PTS Ntr , which controls the activity of K ؉ transporters in response to unknown signals. This is achieved by binding of dephosphorylated EIIA Ntr to other proteins. We generated LicT mutants that were controlled either negatively by HPr or positively by NPr and were suitable bio-bricks, in order to monitor or to couple gene expression to the phosphorylation states of these two proteins. With the aid of these tools, we identified the stringent starvation protein SspA as a regulator of EIIA Ntr phosphorylation, indicating that PTS Ntr represents a stress-related system in E. coli.

ABC Transport Is Inactivated by the PTSNtr under Potassium Limitation in Rhizobium leguminosarum 3841

“…This is in agreement with recent results in E. coli where non-phosphorylatable PtsN is required to activate PhoR [19]. However, in a physiological condition where E. coli PtsN is shown to be fully phosphorylated [41], both phosphorylatable and non-phosphorylatable PtsN versions were equally able to activate PhoR mediated transcriptional responses when expressed from a plasmid [19]. This clearly shows that absolute levels and relative degree of phosphorylation are essential for PtsN function.…”

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.…”

Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16

“…As a common theme, exclusively non-phosphorylated EIIA Ntr is competent in protein-protein interaction. Generally, the phosphorylated form of EIIA Ntr prevails when cells grow in rich media (Bahr et al, 2011;Jahn et al, 2013;Pflüger & de Lorenzo, 2007). In contrast to the canonical EI, EI Ntr contains a GAF signalling domain, which modulates autophosphorylation of EI Ntr and thereby the phosphorylation state of PTS Ntr (Lee et al, 2013).…”