Cross-Talk between the Canonical and the Nitrogen-Related Phosphotransferase Systems Modulates Synthesis of the KdpFABC Potassium Transporter in Escherichia coli.

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).

The phosphotransferase protein EIIA(Ntr) modulates the phosphate starvation response through interaction with histidine kinase PhoR in Escherichia coli.

Many Proteobacteria possess the paralogous PTS(Ntr), in addition to the sugar transport phosphotransferase system (PTS). In the PTS(Ntr) phosphoryl-groups are transferred from phosphoenolpyruvate to protein EIIA(Ntr) via the phosphotransferases EI(Ntr) and NPr. The PTS(Ntr) has been implicated in regulation of diverse physiological processes. In Escherichia coli, the PTS(Ntr) plays a role in potassium homeostasis. In particular, EIIA(Ntr) binds to and stimulates activity of a two-component histidine kinase (KdpD) resulting in increased expression of the genes encoding the high-affinity K(+) transporter KdpFABC. Here, we show that the phosphate (pho) regulon is likewise modulated by PTS(Ntr). The pho regulon, which comprises more than 30 genes, is activated by the two-component system PhoR/PhoB under conditions of phosphate starvation. Mutants lacking EIIA(Ntr) are unable to fully activate the pho genes and exhibit a growth delay upon adaptation to phosphate limitation. In contrast, pho expression is increased above the wild-type level in mutants deficient for EIIA(Ntr) phosphorylation suggesting that non-phosphorylated EIIA(Ntr) modulates pho. Protein interaction analyses reveal binding of EIIA(Ntr) to histidine kinase PhoR. This interaction increases the amount of phosphorylated response regulator PhoB. Thus, EIIA(Ntr) is an accessory protein that modulates the activities of two distinct sensor kinases, KdpD and PhoR, in E. coli.

Insight into bacterial phosphotransferase system-mediated signaling by interspecies transplantation of a transcriptional regulator.

The bacterial sugar:phosphotransferase system (PTS) delivers phosphoryl groups via proteins EI and HPr to the EII sugar transporters. The antitermination protein LicT controls ß-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 ß-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.

Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae.

Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ(54)-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ(70)-promoters overlap the σ(54)-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ(70)-promoter. Thus, transcription of glmY and glmZ is controlled by σ(54) and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription. In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

Stimulation of the potassium sensor KdpD kinase activity by interaction with the phosphotransferase protein IIA(Ntr) in Escherichia coli.

Proteins EI(Ntr), NPr and IIA(Ntr) form a phosphoryl group transfer chain (Ntr-PTS) working in parallel to the phosphoenolpyruvate:carbohydrate phosphotransferase system (transport-PTS) in Escherichia coli. Recently, it was shown that dephosphorylated IIA(Ntr) binds and inhibits TrkA, a low-affinity potassium transporter. Here we report that the Ntr-PTS also regulates expression of the high-affinity K+ transporter KdpFABC, which rescues K+ uptake at limiting K+ concentrations. Transcription initiation at the kdpFABC promoter is positively controlled by the two-component system KdpD/KdpE in response to K+ availability. We found that kdp promoter activity is stimulated by the dephosphorylated form of IIA(Ntr). Two-hybrid data and biochemical analysis revealed that IIA(Ntr) interacts with sensor kinase KdpD and stimulates kinase activity, resulting in increased levels of phosphorylated response regulator KdpE. The data suggest that exclusively dephosphorylated IIA(Ntr) binds and activates KdpD. As there is cross-talk between the Ntr-PTS and the transport-PTS, carbon source utilization affects kdpFABC expression. Expression is enhanced, when cells utilize preferred carbohydrates like glucose, which results in preferential dephosphorylation of the transport-PTS and also of IIA(Ntr). Taken together, the data show that the Ntr-PTS has an important role in maintaining K+ homeostasis and links K+ uptake to carbohydrate metabolism.