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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.

SUBMITTER: Bahr T

PROVIDER: S-EPMC3133047 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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

Bahr Thomas T Lüttmann Denise D März Walter W Rak Bodo B Görke Boris B

Journal of bacteriology 20110218 8

The bacterial sugar:phosphotransferase system (PTS) delivers phosphoryl groups via proteins EI and HPr to the EII sugar transporters. The antitermination protein LicT controls β-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 β-glucoside transporter ...[more]

PMID: 21335451

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Project description:BACKGROUND: The major uptake system responsible for the transport of fructose, glucose, and sucrose in Corynebacterium glutamicum ATCC 13032 is the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The genes encoding PTS components, namely ptsI, ptsH, and ptsF belong to the fructose-PTS gene cluster, whereas ptsG and ptsS are located in two separate regions of the C. glutamicum genome. Due to the localization within and adjacent to the fructose-PTS gene cluster, two genes coding for DeoR-type transcriptional regulators, cg2118 and sugR, are putative candidates involved in the transcriptional regulation of the fructose-PTS cluster genes. RESULTS: Four transcripts of the extended fructose-PTS gene cluster that comprise the genes sugR-cg2116, ptsI, cg2118-fruK-ptsF, and ptsH, respectively, were characterized. In addition, it was shown that transcription of the fructose-PTS gene cluster is enhanced during growth on glucose or fructose when compared to acetate. Subsequently, the two genes sugR and cg2118 encoding for DeoR-type regulators were mutated and PTS gene transcription was found to be strongly enhanced in the presence of acetate only in the sugR deletion mutant. The SugR regulon was further characterized by microarray hybridizations using the sugR mutant and its parental strain, revealing that also the PTS genes ptsG and ptsS belong to this regulon. Binding of purified SugR repressor protein to a 21 bp sequence identified the SugR binding site as an AC-rich motif. The two experimentally identified SugR binding sites in the fructose-PTS gene cluster are located within or downstream of the mapped promoters, typical for transcriptional repressors. Effector studies using electrophoretic mobility shift assays (EMSA) revealed the fructose PTS-specific metabolite fructose-1-phosphate (F-1-P) as a highly efficient, negative effector of the SugR repressor, acting in the micromolar range. Beside F-1-P, other sugar-phosphates like fructose-1,6-bisphosphate (F-1,6-P) and glucose-6-phosphate (G-6-P) also negatively affect SugR-binding, but in millimolar concentrations. CONCLUSION: In C. glutamicum ATCC 13032 the DeoR-type regulator SugR acts as a pleiotropic transcriptional repressor of all described PTS genes. Thus, in contrast to most DeoR-type repressors described, SugR is able to act also on the transcription of the distantly located genes ptsG and ptsS of C. glutamicum. Transcriptional repression of the fructose-PTS gene cluster is observed during growth on acetate and transcription is derepressed in the presence of the PTS sugars glucose and fructose. This derepression of the fructose-PTS gene cluster is mainly modulated by the negative effector F-1-P, but reduced sensitivity to the other effectors, F-1,6-P or G-6-P might cause differential transcriptional regulation of genes of the general part of the PTS (ptsI, ptsH) and associated genes encoding sugar-specific functions (ptsF, ptsG, ptsS).

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

Publications

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

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