In Vitro Reconstitution of Catabolite Repression in Escherichia coli

A widely accepted model for catabolite repression posits that phospho-IIAGlc of the bacterial phosphotransferase system activates adenylyl cyclase (AC) activity. For many years, attempts to observe such regulatory properties of AC in vitro have been unsuccessful. To further study the regulation, AC...

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Bibliographic Details
Published in:The Journal of biological chemistry 2006-03, Vol.281 (10), p.6448-6454
Main Authors: Park, Young-Ha, Lee, Byeong R., Seok, Yeong-Jae, Peterkofsky, Alan
Format: Article
Language:English
Subjects:
Adenylyl Cyclase Inhibitors
Adenylyl Cyclases - physiology
Cell Membrane - enzymology
Cyclic AMP - metabolism
Escherichia coli
Escherichia coli - enzymology
Escherichia coli Proteins - antagonists & inhibitors
Escherichia coli Proteins - physiology
In Vitro Techniques
Phosphoenolpyruvate Sugar Phosphotransferase System - physiology
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
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Summary:A widely accepted model for catabolite repression posits that phospho-IIAGlc of the bacterial phosphotransferase system activates adenylyl cyclase (AC) activity. For many years, attempts to observe such regulatory properties of AC in vitro have been unsuccessful. To further study the regulation, AC was produced fused to the transmembrane segments of the serine chemoreceptor Tsr. Cells harboring Tsr-AC and normal AC, expressed from the cya promoter on a low copy number vector, exhibit similar behavior with respect to elevation of cAMP levels resulting from deletion of crp, expressing the catabolite regulatory protein. Membrane-bound Tsr-AC exhibits activity comparable with the native form of AC. Tsr-AC binds IIAGlc specifically, regardless of its phosphorylation state, but not the two general phosphotransferase system proteins, enzyme I and HPr; IIAGlc binding is localized to the C-terminal region of AC. Binding to membranes of either dephospho- or phospho-IIAGlc has no effect on AC activity. However, in the presence of an Escherichia coli extract, P-IIAGlc, but not IIAGlc, stimulates AC activity. Based on these findings of a direct interaction of IIAGlc with AC, but activity regulation only in the presence of E. coli extract, a revised model for AC activity regulation is proposed.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M512672200