Outer membrane of Salmonella typhimurium: accessibility of phospholipid head groups to phospholipase C and cyanogen bromide activated dextran in the external medium

191

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Lipopolysaccharide (LPS) is the major glycolipid that is present in the outer membranes (OMs) of most Gram-negative bacteria. LPS molecules are assembled with divalent metal cations in the outer leaflet of the OM to form an impervious layer that prevents toxic compounds from entering the cell. For most Gram-negative bacteria, LPS is essential for growth. In Escherichia coli, eight essential proteins have been identified to function in the proper assembly of LPS following its biosynthesis. This assembly process involves release of LPS from the inner membrane (IM), transport across the periplasm, and insertion into the outer leaflet of the OM. Here, we describe the biochemical characterization of the two-protein complex consisting of LptD and LptE that is responsible for the assembly of LPS at the cell surface. We can overexpress and purify LptD and LptE as a stable complex in a 1∶1 stoichiometry. LptD contains a soluble N-terminal domain and a C-terminal transmembrane domain. LptE stabilizes LptD by interacting strongly with the C-terminal domain of LptD. We also demonstrate that LptE binds LPS specifically and may serve as a substrate recognition site at the OM. T he OM of Gram-negative bacteria is a unique asymmetric lipid bilayer in which the outer leaflet is composed almost entirely of LPS and the inner leaflet of phospholipids (PL) (Fig. 1) (1, 2). Building and maintaining this asymmetric bilayer is a challenge for the cell because the OM is located outside the cytoplasm, in an environment that lacks an obvious energy source such as ATP. LPS and PL are synthesized at the cytoplasmic face of the IM whereas lipoproteins and integral membrane proteins are synthesized in the cytoplasm; all these OM components must be transported across the IM and the aqueous periplasmic compartment to be assembled into the OM (3, 4). In the case of LPS assembly, the molecule must ultimately reach the outer leaflet of the OM (Fig.

Section: Resultsmentioning

confidence: 99%

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

Chng

Ruiz

Chimalakonda

et al. 2010

191

247

“…*This Direct Submission article had a prearranged editor. 1 To whom correspondence should be addressed. E-mail: kahne@chemistry.harvard.edu.…”

Section: Resultsmentioning

confidence: 99%

“…lipopolysaccharide assembly | outer membrane protein complex T he outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer (1)(2)(3), with an inner leaflet composed of phospholipids and an outer leaflet consisting mainly of lipopolysaccharide (LPS). In Escherichia coli, the LPS molecule typically contains six fatty acyl chains and as many as several hundred sugars.…”

mentioning

confidence: 99%

The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel

Freinkman

Chng

Kahne

2011

164

181

The cell surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS). This glycolipid is found exclusively in the outer leaflet of the asymmetric outer membrane (OM), where it forms a barrier to the entry of toxic hydrophobic molecules into the cell. LPS typically contains six fatty acyl chains and up to several hundred sugar residues. It is biosynthesized in the cytosol and must then be transported across two membranes and an aqueous intermembrane space to the cell surface. These processes are required for the viability of most Gram-negative organisms. The integral membrane β-barrel LptD and the lipoprotein LptE form an essential complex in the OM, which is necessary for LPS assembly. It is not known how this complex translocates large, amphipathic LPS molecules across the OM to the outer leaflet. Here, we show that LptE resides within the LptD β-barrel both in vitro and in vivo. LptD/E associate via an extensive interface; in one specific interaction, LptE contacts a predicted extracellular loop of LptD through the lumen of the β-barrel. Disrupting this interaction site compromises the biogenesis of LptD. This unprecedented two-protein plug-and-barrel architecture suggests how LptD/E can insert LPS from the periplasm directly into the outer leaflet of the OM to establish the asymmetry of the bilayer.lipopolysaccharide assembly | outer membrane protein complex

“…The IM is composed of phospholipids, integral transmembrane (TM) proteins that span the IM with ␣-helical TM domains, and lipoproteins (3,4). In contrast, the OM is typically an asymmetric lipid bilayer where the inner leaflet is composed of phospholipids and the outer leaflet is composed mainly of lipopolysaccharide (LPS) (5). In addition, the OM contains lipoproteins and integral outer membrane proteins (OMPs), most of which span the OM via antiparallel ␤-sheets that fold into ␤-barrels (4,6).…”

mentioning

confidence: 99%

Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli

Ruiz

Gronenberg

Kahne

et al. 2008

241

277

The outer membrane (OM) of most Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet. LPS, or endotoxin, is a molecule of important biological activities. In the host, LPS elicits a potent immune response, while in the bacterium, it plays a crucial role by establishing a barrier to limit entry of hydrophobic molecules. Before LPS is assembled at the OM, it must be synthesized at the inner membrane (IM) and transported across the aqueous periplasmic compartment. Much is known about the biosynthesis of LPS but, until recently, little was known about its transport and assembly. We applied a reductionist bioinformatic approach that takes advantage of the small size of the proteome of the Gram-negative endosymbiont Blochmannia floridanus to search for novel factors involved in OM biogenesis. This led to the discovery of two essential Escherichia coli IM proteins of unknown function, YjgP and YjgQ, which are required for the transport of LPS to the cell surface. We propose that these two proteins, which we have renamed LptF and LptG, respectively, are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM.ABC transporter ͉ bioinformatics ͉ endosymbiont ͉ membrane biogenesis T he hallmark of Gram-negative bacteria is the presence of two extracytoplasmic membranes: the inner and outer membranes. The inner membrane (IM), which surrounds the cytoplasm, is separated from the outer membrane (OM) by an aqueous compartment known as the periplasm (1, 2). The IM is composed of phospholipids, integral transmembrane (TM) proteins that span the IM with ␣-helical TM domains, and lipoproteins (3, 4). In contrast, the OM is typically an asymmetric lipid bilayer where the inner leaflet is composed of phospholipids and the outer leaflet is composed mainly of lipopolysaccharide (LPS) (5). In addition, the OM contains lipoproteins and integral outer membrane proteins (OMPs), most of which span the OM via antiparallel ␤-sheets that fold into ␤-barrels (4, 6). Although some Gram-negative bacteria lack LPS and in others LPS is not essential (7), LPS is essential in Escherichia coli, Salmonella, and probably many other bacteria.In E. coli, the main function of the OM is to serve as a selective permeability barrier against many toxic chemicals, such as detergents and antibiotics (1). Porin proteins in the OM control permeability to hydrophilic molecules, but unlike typical phospholipid bilayers, the OM is quite impermeable to hydrophobic molecules, mainly because of LPS (1). For the OM to serve as a barrier, all OM components must be assembled properly. None of the components of the OM are synthesized in situ, so they must be transported to the OM from their site of synthesis. All OMPs and lipoproteins are made in the cytoplasm and cross the IM through the Sec translocon (3). After their signal sequence is removed, OMPs are thought to travel across the periplasm aided by chaperones that deliver them to the OM Bam complex (␤-barrel a...