In Vitro Reconstitution of Catabolite Repression in Escherichia coli

Young-Ha Park,Byeong R Lee,Yeong-Jae Seok,Alan Peterkofsky

doi:10.1074/jbc.m512672200

Abstract

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.

Highlights

In Escherichia coli, cAMP, produced by adenylyl cyclase (AC),5 is an important regulatory molecule, essential for controlling the expression of numerous operons
PDIA(tsr-cya)—As a first step in developing the methodology, it was necessary to determine whether the tethering of AC to the membrane resulted in a protein that was still capable of regulation in the same way that normal AC is regulated
The expression of the Tsr-AC fusion protein is under the control of the normal cya promoter; the low level expression of the gene in this construct avoids the toxicity associated with high levels of cAMP [17, 18]

Summary

Introduction

In Escherichia coli, cAMP, produced by adenylyl cyclase (AC), is an important regulatory molecule, essential for controlling the expression of numerous operons. One approach to allow a further understanding of the mechanism by which AC is regulated has involved the use of permeable cells In this case, exposure of the permeable cells to glucose results in inhibition of AC activity [5]. Liberman et al [6] demonstrated that unphosphorylated IIAGlc resulted in a Pi-dependent inhibition of AC in permeable cells These data suggested that dephosphorylated IIAGlc can interact with and influence the activity of AC. The partially purified AC or diluted crude extracts showed no effect of added PTS proteins, relatively concentrated extracts showed a variety of effects characteristic of AC activity in permeable cells These experiments suggested that at least one other uncharacterized factor was required for the effective coupling of PTS proteins to AC. It has been repeatedly speculated elsewhere [8, 9] that other unknown factors could be involved in the regulation of AC activity

Methods

Results

Conclusion