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Glucose-6-phosphate-dependent phosphoryl flow through the Uhp two-component regulatory system
D T Verhamme1, J C Arents, P W Postma
1Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands.
Microbiology (Reading, England)
|December 12, 2001
Summary
The UhpABC system in E. coli regulates sugar-phosphate transporter (uhpT) expression. Glucose 6-phosphate (G6P) triggers a signaling cascade involving UhpC, UhpB, and UhpA, controlling uhpT transcription.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The UhpABC system in Escherichia coli controls the expression of the UhpT sugar-phosphate transporter.
- This regulation occurs at the transcriptional level, involving a complex signaling cascade.
Purpose of the Study:
- To demonstrate transmembrane signal transfer within the UhpABC system using an isolated membrane vesicle setup.
- To investigate the phosphorylation and dephosphorylation dynamics of UhpB and UhpA in response to glucose 6-phosphate (G6P).
Main Methods:
- Utilized UhpBC-enriched membrane vesicles from E. coli.
- Employed [gamma-(32)P]ATP to monitor protein phosphorylation.
- Assessed UhpB autophosphorylation and UhpA phosphorylation/dephosphorylation in response to intra-vesicular G6P.
Main Results:
- UhpB autophosphorylation was stimulated by intra-vesicular G6P sensing via UhpC.
- Addition of UhpA led to rapid and transient UhpA phosphorylation.
- G6P-induced UhpBC vesicles promoted rapid UhpA dephosphorylation, indicating dominant UhpB phosphatase activity.
Conclusions:
- Transmembrane signal transfer in the UhpABC system was demonstrated in vitro.
- UhpB exhibits both kinase and phosphatase activities, with phosphatase activity dominating in the G6P-activated state.
- Low in vivo P~UhpA levels or stabilization by uhpT promoter DNA may be necessary for sustained uhpT transcription induction.