%0 Journal Article %A Verhamme, Daniël T. %A Arents, Jos C. %A Postma, Pieter W. %A Crielaard, Wim %A Hellingwerf, Klaas J. %T Glucose-6-phosphate-dependent phosphoryl flow through the Uhp two-component regulatory system %D 2001 %J Microbiology, %V 147 %N 12 %P 3345-3352 %@ 1465-2080 %R https://doi.org/10.1099/00221287-147-12-3345 %K transmembrane signalling %K HPK, histidine-protein kinase %K RR, response-regulator %K kinase/phosphatase state %K G6P, glucose 6-phosphate %K RSO, right-side-out %K ISO, inside-out %I Microbiology Society, %X Expression of the UhpT sugar-phosphate transporter in Escherichia coli is regulated at the transcriptional level via the UhpABC signalling cascade. Sensing of extracellular glucose 6-phosphate (G6P), by membrane-bound UhpC, modulates a second membrane-bound protein, UhpB, resulting in autophosphorylation of a conserved histidine residue in the cytoplasmic (transmitter) domain of the latter. Subsequently, this phosphoryl group is transferred to a conserved aspartate residue in the response-regulator UhpA, which then initiates uhpT transcription, via binding to the uhpT promoter region. This study demonstrates the hypothesized transmembrane signal transfer in an ISO membrane set-up, i.e. in a suspension of UhpBC-enriched membrane vesicles, UhpB autophosphorylation is stimulated, in the presence of [γ-32P]ATP, upon intra-vesicular sensing of G6P by UhpC. Subsequently, upon addition of UhpA, very rapid and transient UhpA phosphorylation takes place. When P∼UhpA is added to G6P-induced UhpBC-enriched membrane vesicles, rapid UhpA dephosphorylation occurs. So, in the G6P-activated state, UhpB phosphatase activity dominates over kinase activity, even in the presence of saturating amounts of G6P. This may imply that maximal in vivo P∼UhpA levels are low and/or that, to keep sufficient P∼UhpA accumulated to induce uhpT transcription, the uhpT promoter DNA itself is involved in stabilization/sequestration of P∼UhpA. %U https://www.microbiologyresearch.org/content/journal/micro/10.1099/00221287-147-12-3345