Deletion of the Heptosyltransferase Genes rfaC and rfaF in Escherichia Coli K‐12 Results in an Re‐Type Lipopolysaccharide with a High Degree of 2‐Aminoethanol Phosphate Substitution

Brabetz, Werner; Müller‐Loennies, Sven; Holst, Otto; Brade, Helmut

doi:10.1111/j.1432-1033.1997.00716.x

Cited by 116 publications

(167 citation statements)

References 57 publications

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“…We also found minor peaks (m/z 1,921-1,940; Fig. 3C and data not shown) indicative of additional modifications that could correspond to phosphoethanolamine-or aminodeoxypentose-modified lipid A, molecules that appear when the OM is damaged (39,47). Therefore, even under inducing conditions, our depletion strains have slight defects in LPS biogenesis.…”

Section: Depletion Of Lptf And/or Lptg Leads To Altered Lps But Does Notmentioning

confidence: 80%

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

Proc. Natl. Acad. Sci. U.S.A.

241

277

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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...

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Section: Depletion Of Lptf And/or Lptg Leads To Altered Lps But Does Notmentioning

confidence: 80%

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

Proc. Natl. Acad. Sci. U.S.A.

241

277

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“…Many pathogens, such as strains of Pseudomonas aeruginosa in cystic fibrosis patients (29,30), now are resistant to commercially available antibiotics. Therefore it is necessary to develop new antibacterial agents that target previously unexploited systems, such as the enzymes that assemble the lipid A component of lipopolysaccharide (2,8,9,(31)(32)(33)(34)(35). The cytosolic acyltransferase LpxA catalyzes the first step of the lipid A pathway ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide

Williams

Immormino

Gewirth

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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UDP-GlcNAc acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein (ACP) to the glucosamine 3-OH group of UDP-GlcNAc. LpxA is essential for the growth of Escherichia coli and related Gram-negative bacteria. The crystal structure of the E. coli LpxA homotrimer, determined previously at 2.6 Å in the absence of substrates or inhibitors, revealed that LpxA contains an unusual, left-handed parallel ␤-helix fold. We now present the crystal structure at 1.8 Å resolution of E. coli LpxA in a complex with a pentadecapeptide, peptide 920. Three peptides, each of which adopts a ␤-hairpin conformation, are bound per LpxA trimer. The peptides are located at the interfaces of adjacent subunits in the vicinity of the three active sites. Each peptide interacts with residues from both adjacent subunits. Peptide 920 is a potent inhibitor of E. coli LpxA (Ki ‫؍‬ 50 nM). It is competitive with respect to acyl-ACP but not UDP-GlcNAc. The compact ␤-turn structure of peptide 920 bound to LpxA may open previously uncharacterized approaches to the rational design of LpxA inhibitors with antibiotic activity.antibiotic ͉ crystal structure ͉ Escherichia coli ͉ inhibitor ͉ outer membrane L ipopolysaccharide constitutes the outer monolayer of the outer membrane in Gram-negative bacteria (1-3). Lipopolysaccharide maintains the integrity of the outer membrane, which functions as a barrier to molecules Ͼ500 Da (4). Lipid A (endotoxin) is the hydrophobic moiety that anchors lipopolysaccharide into the outer membrane (1, 2). It is a potent activator of the human innate immune system via Toll-like receptor 4 (TLR4) (5-7). The minimal lipopolysaccharide required for growth usually consists of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues attached to lipid A ( Fig. 1; ref. 2). Because lipid A biosynthetic enzymes have no mammalian counterparts, they are attractive targets for the design of new antibiotics (10, 11).Kdo 2 -lipid A is synthesized by a conserved pathway consisting of nine enzymes (1, 2). LpxA catalyzes the first step (Fig. 1), the thermodynamically unfavorable 3-O-acylation of UDP-GlcNAc (K eq ϭ 0.01) (12-14). The crystal structure of Escherichia coli LpxA, previously determined at 2.6 Å (15), revealed that the enzyme is a homotrimer. It adopts a distinctive, left-handed parallel ␤-helix fold ( Fig. 2 A and B), which is specified by the presence of 24 complete and six partial hexad repeats (15). Three contiguous hexad repeats (18-aa residues) fold into one coil of the ␤-helix (Fig. 2 A and B). Many other bacterial acyl and acetyl transferases later were shown to possess this fold (17)(18)(19)(20)(21)(22).Structures of LpxA have not been solved in the presence of substrates or inhibitors. Mutagenesis suggests that the active site is located in a large cleft between adjacent subunits (8, 23). This region contains several conserved basic residues (K76, H122, H125, H144, H160, and R204) (23). H125 (Fig. 2 C and D) may be the catalytic ...

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“…Isolation of Kdo 2 -Lipid A-WBB06 cells (27) were grown overnight at 37°C to A 600 ϳ3.5 in 2 liters of LB broth adjusted to pH 7.0 and supplemented with 12 g/ml tetracycline. Cells were harvested by centrifugation at 6,000 ϫ g for 10 min, washed with an equal volume of phosphate-buffered saline (28), and resuspended in 160 ml of phosphate-buffered saline.…”

Section: Methodsmentioning

confidence: 99%

ATPase Activity of the MsbA Lipid Flippase of Escherichia coli

Doerrler¹,

Raetz

2002

Journal of Biological Chemistry

202

160

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Deletion of the Heptosyltransferase Genes rfaC and rfaF in Escherichia Coli K‐12 Results in an Re‐Type Lipopolysaccharide with a High Degree of 2‐Aminoethanol Phosphate Substitution

Cited by 116 publications

References 57 publications

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

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

Structure of UDP-N-acetylglucosamine acyltransferase with a bound antibacterial pentadecapeptide

ATPase Activity of the MsbA Lipid Flippase of Escherichia coli

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