HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 12, 8583-8593, March 23, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/12/8583    most recent M605785200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fleischer, R. Articles by Hunke, S. Search for Related Content PubMed PubMed Citation Articles by Fleischer, R. Articles by Hunke, S. Social Bookmarking                      What's this?

Purification, Reconstitution, and Characterization of the CpxRAP Envelope Stress System of Escherichia coli^*

Rebecca Fleischer, Ralf Heermann, Kirsten Jung, and Sabine Hunke¹

From the Institut für Biologie, Abteilung Physiologie der Mikroorganismen, Humboldt Universität zu Berlin, D-10115 Berlin, Germany and the Department Biologie I, Bereich Mikrobiologie, Ludwig-Maximilians-Universität, Maria-Ward-Strasse 1a, D-80638 München, Germany

In Escherichia coli the Cpx sensor regulator system senses different kinds of envelope stress and responds by triggering the expression of periplasmic folding factors and proteases. It consists of the membrane-anchored sensor kinase CpxA, the response regulator CpxR, and the periplasmic protein CpxP. The Cpx pathway is induced in vivo by a variety of signals including pH variation, osmotic stress, and misfolded envelope proteins and is inhibited by overproduced CpxP. Because it is not clear how the Cpx pathway is able to recognize and correspond to so many different signals we overproduced, solubilized, purified, and incorporated the complete membrane-integral CpxA protein into proteoliposomes to analyze its biochemical properties in more detail. Autokinase and phosphotransfer activities of the reconstituted CpxA-His₆ protein were stimulated by KCl. NaCl also stimulated the activities but to a lesser extent. Other osmotic active solutes as glycine betaine, sucrose, and proline had no effect. The system was further characterized by testing for susceptibility to sensor kinase inhibitors. Among these, Closantel inhibited the activities of solubilized but not of the reconstituted CpxA-His₆ protein. We further analyzed the effect of CpxP on CpxA activities. Purified tagless CpxP protein reduced the phosphorylation status of CpxA to 50% but had no effect on CpxA phosphotransfer or phosphatase activities. As the in vitro system excludes the involvement of other factors our finding is the first biochemical evidence for direct protein-protein interaction between the sensor kinase CpxA and the periplasmic protein CpxP resulting in a down-regulation of the autokinase activity of CpxA.

Received for publication, June 16, 2006 , and in revised form, October 16, 2006.

^* This work was supported by grants from the Deutsche Forschungsgemeinschaft (Hu1011/1-1; to S. H. and R. F.) and (JU270/3-3; to K. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Physiologie der Mikroorganismen, Chausseestr. 117, D-10115 Berlin, Germany. Tel.: 0049-0-30-2093-8122; Fax: 0049-0-30-2093-8126; E-mail: sabine.hunke{at}rz.hu-berlin.de.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. J. Wolfe, N. Parikh, B. P. Lima, and B. Zemaitaitis Signal Integration by the Two-Component Signal Transduction Response Regulator CpxR J. Bacteriol., April 1, 2008; 190(7): 2314 - 2322. [Abstract] [Full Text] [PDF]

				D. M. MacRitchie, J. D. Ward, A. Z. Nevesinjac, and T. L. Raivio Activation of the Cpx Envelope Stress Response Down-Regulates Expression of Several Locus of Enterocyte Effacement-Encoded Genes in Enteropathogenic Escherichia coli Infect. Immun., April 1, 2008; 76(4): 1465 - 1475. [Abstract] [Full Text] [PDF]

				Y. Eguchi, J. Itou, M. Yamane, R. Demizu, F. Yamato, A. Okada, H. Mori, A. Kato, and R. Utsumi B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli PNAS, November 20, 2007; 104(47): 18712 - 18717. [Abstract] [Full Text] [PDF]

				M. Miot and J.-M. Betton Optimization of the inefficient translation initiation region of the cpxP gene from Escherichia coli Protein Sci., November 1, 2007; 16(11): 2445 - 2453. [Abstract] [Full Text] [PDF]

				N. Moker, P. Reihlen, R. Kramer, and S. Morbach Osmosensing Properties of the Histidine Protein Kinase MtrB from Corynebacterium glutamicum J. Biol. Chem., September 21, 2007; 282(38): 27666 - 27677. [Abstract] [Full Text] [PDF]