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J. Biol. Chem., Vol. 280, Issue 6, 4504-4509, February 11, 2005

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Lipopolysaccharide Transport to the Bacterial Outer Membrane in Spheroplasts^*

Boris Tefsen, Jeroen Geurtsen, Frank Beckers, Jan Tommassen, and Hans de Cock

From the Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner membrane to the outer membrane is largely unknown. Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins. Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released. We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the outer membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner membrane. The outer membrane localization of the LPS was confirmed by its modification by the outer membrane enzyme CrcA (PagP). We conclude that a substantial amount of LPS was translocated to the outer membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two membranes. In contrast to LPS, de novo synthesized phospholipids were not transported to the outer membrane in spheroplasts. Apparently, LPS and phospholipids have different requirements for their transport to the outer membrane.

Received for publication, August 12, 2004 , and in revised form, November 26, 2004.

^* This work was supported by the Research Council for Earth and Life Sciences (ALW) with financial aid from the Netherlands Organization for Scientific Research (NWO). 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.

To whom correspondence should be addressed. Tel.: 31-30-2532267; Fax: 31-30-2513655; E-mail: J.J.P.A.deCock{at}bio.uu.nl.

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