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The outer membrane of gram-negative bacteria is a barrier to many antibiotics and host defense factors (14). This barrier function is due mainly to structural features of the lipopolysaccharide (LPS) molecules that make up the outer leaflet of the outer membrane bilayer. In Escherichia coli, the LPS molecule is conceptually divided into three parts (Fig. 1). Lipid A, the hydrophobic membrane anchor, is responsible for the endotoxic properties of LPS. Attached to lipid A is the core region, a phosphorylated oligosaccharide composed of 10 to 15 sugar molecules, which serves as the attachment site for the O antigen. Finally, the O antigen is a structurally variable polysaccharide made up of repeating oligomeric units. A critical feature of E. coli LPS is the presence of phosphoryl substituents on the heptose residues of the LPS core (Fig. 1). These phosphate-containing substituents are essential to membrane stability, likely because their negative charge allows neighboring LPS molecules to be cross-linked by divalent cations (9, 14).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Structure of the LPS from E. coli strains with an R1-type core. Core residues are designated by sugar abbreviation and number to facilitate identification. Abbreviations are as follows: GlcN, D-glucosamine; Hep, L-glycero-D-manno-heptose; P, phosphate; EtNP, 2-aminoethyl phosphate; Glc, D-glucose; and Gal, D-galactose. All sugars are in the pyranose configuration, and the linkages are  unless otherwise indicated. The assignment of function to genes encoding core glycosyltransferases and phosphotransferases has been reported previously (2, 3, 7, 19). Of particular note, the waaG gene product (indicated by an asterisk) is the glucosyltransferase for GlcI.

The genes involved in LPS core phosphorylation have recently been characterized; the waaP gene product transfers phosphate to HepI, while waaY encodes the phosphotransferase that acts upon HepII (Fig. 1) (19). Of note, WaaY cannot function until after WaaP does, so that mutation of waaP alone is enough to completely eliminate core heptose phosphorylation. The waaP and waaY genes are located in the middle operon of the three operons in the waa (formerly rfa) locus on the chromosome; these operons encode all of the glycosyltransferases necessary for core elongation (4). Precise functions have been assigned to these glycosyltransferases by determining the core structures resulting from defined nonpolar insertions (Fig. 1) (3, 19).

In this study, the set of defined mutants used to determine core glycosyltransferase function was subjected to antimicrobial sensitivity testing with sodium dodecyl sulfate (SDS) and novobiocin as a measure of outer membrane stability. As expected, the mutants defective in core phosphorylation showed an increased susceptibility to these compounds. However, a surprising observation from this screening was that the defined E. coli waaG mutant (with LPS truncated after HepII [Fig. 1]) also appeared to have a compromised outer membrane barrier function. Therefore, the underlying mechanism for the waaG phenotype was investigated by a variety of structural methods.

Bacterial strains. E. coli F470 is the prototype strain for studies of the R1-type core oligosaccharide (11), and the structure of its complete core is known (6, 16). The R1 prototype was used in these and prior studies because it is the most prevalent among clinical and natural isolates of E. coli (1). Derivatives CWG303 (F470 with a waaG::aacC1 insertion), CWG308 (F470 with a waaO::aacC1 insertion), CWG296 (F470 with a waaP::aacC1 insertion), CWG297 (F470 with a waaQ::aacC1 insertion), and CWG312 (F470 with a waaY::aacC1 insertion) have been described previously (3, 19). The aacC1 cassette contains no transcriptional stop sites, and the absence of translational polarity was verified by complementation to the wild-type phenotype with the appropriate gene in trans.

Novobiocin and SDS sensitivity testing. To determine the MICs of novobiocin and SDS for each core mutant, twofold serial dilutions of novobiocin (from 200 to 1.6 µg/ml) and SDS (from 200 to 0.1 mg/ml) were made in 5-ml tubes of Luria-Bertani broth. Each series of tubes was then inoculated from an overnight culture and incubated with shaking at 37°C. Growth was scored as positive if the culture was visibly turbid (i.e., optical density at 600 nm of >0.2) after 8 h. Trials were performed in triplicate and repeated on two separate occasions.

LPS purification. LPS was obtained by a modification of the phenol-water cell extraction method (17). Wet cells (300 g, containing approximately 20% dry material) were mixed with 240 ml of phenol and stirred for 30 min at 70°C. Instead of separating the phenol and water phases as described previously, the entire extraction mixture was diluted with 500 ml of water and then dialyzed (against water) for 1 week. LPS was subsequently isolated by centrifugation, with the remainder of the published phenol-water extraction protocol being essentially unchanged.

LPS deacylation, and analysis of resulting oligosaccharides (OS-1 and OS-2). LPS (100 mg) was deacylated and purified as described previously (15). Two major products, OS-1 and OS-2, were observed in a ratio of approximately 3:2 (data not shown). These oligosaccharides were analyzed by nuclear magnetic resonance (NMR) spectroscopy. The two products had the same carbohydrate skeleton, with the only difference being the presence of an additional phosphate group at O-4 of Hep residue E in OS-2 (Fig. 2). The identities of the monosaccharides were established on the basis of chemical shifts and vicinal proton coupling constants, which were in agreement with expected values based on the published structure of the F470 core (6, 16). The sequence of the monomeric components was determined by using nuclear Overhauser effect (NOE) data. NOE correlations between protons B1A6, F1E3, E1C5, E1C7, C3D6, and C3D8 were observed (data not shown), again corresponding to the sequence in Fig. 2. The positions of the phosphate residues were determined by using ¹H-³¹P NMR correlations (data not shown). The electrospray mass spectra of OS-1 and OS-2 were also recorded (using a Micromass Quattro spectrometer). These spectra contained major peaks at m/z 1,325.1 and 1,405.1 again in agreement with the structures presented in Fig. 2. (The 80-mass-unit difference is due to the absence or presence of phosphate.)

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Deduced structure of the LPS from CWG303 (waaG). The two major oligosaccharides observed after complete deacylation of CWG303 LPS differ only in the presence or absence of a phosphate (P) residue on Hep residue E (R = OH in OS-1; R = P in OS-2). The major oligosaccharides after mild acid hydrolysis of CWG303 LPS again differ only by phosphorylation on Hep residue E (R = OH in OS-3; R = P or 2-aminoethyl diphosphate in OS-4). The P substitution on Hep residue E (HepI) occurs in about 40% of LPS molecules, with 30% of these P residues being further modified by PEtN.

Mild acid hydrolysis of LPS, and analysis of resulting oligosaccharides (OS-3 and OS-4). LPS (100 mg) was hydrolyzed in 2% acetic acid (which cleaves the acid-labile ketosidic linkage between 3-deoxy-D-manno-oct-2-ulosonic acid [Kdo] and lipid A) and purified as described previously (15). Two main products, OS-3 and OS-4, were observed, both being mixtures of several structures differing by Kdo derivatives. Again, the resulting oligosaccharides were analyzed by NMR and electrospray mass spectrometry. The NMR spectra of OS-3 and OS-4 were partially interpreted (data not shown) and were found to correspond to the structures presented in Fig. 2. OS-3 and OS-4 also differ in the absence or presence of phosphate at Hep residue E. However, about 30% of the OS-4 product carried an additional 2-aminoethyl phosphate (PEtN) substitution (measured by integration of PEtN H-2 and the sum of both Hep H-1 ¹H NMR signals). The mass spectrum of OS-3 contained major peaks at m/z 605.3 and 623.2 ([M-1] of the products with anhydro-Kdo-ol and Kdo-ol at the end) and several unidentified minor peaks of higher mass. The OS-4 spectrum had a major peak at m/z 685.1 and a small peak at m/z 703.2 ([M-1] of the products with anhydro-Kdo-ol and Kdo-ol at the end), as well as several minor peaks of higher mass, two of which correspond to the product with an additional PEtN residue (at m/z 808.0 and 826.2). Taken together, the above data indicate the presence of two main LPS structures for the waaG mutant (Fig. 2), differing only by the presence or absence of phosphate on Hep residue E (HepI).

Analysis of outer membrane protein profiles by SDS-PAGE. Supersensitivity to hydrophobic agents is a characteristic often associated with the pleiotropic phenotype termed deep rough, which is exhibited by mutants with severely truncated LPS core regions (i.e., lacking inner-core heptose residues [Fig. 1]). Another characteristic of the deep-rough phenotype is a marked decrease in the protein content of the outer membrane (reviewed in references 4 and 12). Indeed, in the case of deep-rough mutants, a proposed explanation for the increased susceptibility to hydrophobic compounds is that the void left by missing outer membrane proteins is filled by phospholipids, resulting in patches of phospholipid bilayer in the outer membrane (9).

View larger version (104K): [in this window] [in a new window]   FIG. 3.   SDS-PAGE gel (12.5% polyacrylamide) of Sarkosyl-insoluble outer membrane fractions. Lane 1, F470 (parent); lane 2, CWG303 (waaG); lane 3, CWG296 (waaP). Molecular mass markers (in kilodaltons) are indicated to the left of the gel, and major proteins are labeled on the right.

Interpretation of the waaG phenotype. The structural data presented above show that mutation of waaG results in a complete lack of phosphate groups on HepII and in only 40% phosphate substitution at HepI. This corresponds to an 80% total reduction in heptose phosphorylation, given that the modification of HepI and that of HepII are known to be nearly stoichiometric in the wild type (4). The nonstoichiometric modification of HepI phosphate with PEtN appears to be unaffected. The observation that neither CWG303 (waaG) nor CWG296 (waaP) has an altered outer membrane protein profile (Fig. 3) suggests that the main cause of the observed outer membrane instability lies with this change in LPS core structure and not with gross defects in overall outer membrane organization. Also of note, the differential response of the waaP and waaG mutants to SDS and novobiocin is somewhat surprising and must reflect the different modes of action of the two test compounds. It is still unknown why an 80% reduction in core phosphorylation drastically affects the MIC of SDS but not that of novobiocin.