Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (23)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Rick, P. D.
Right arrow Articles by Ho, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rick, P. D.
Right arrow Articles by Ho, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Glycobiology Pages 557-567  

Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis
Introduction
Results
Discussion
Materials and methods
Acknowledgments
References

Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis

Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis

Paul D.Rick3, Gail L.Hubbard, Masahiro Kitaoka1, Hidemi Nagaki1, Takeshi Kinoshita1, Susan Dowd2, Virgil Simplaceanu2, Chien Ho2

Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA and 1Sankyo Co., Ltd., New Lead Research Laboratories, Tokyo, 140, Japan, and the 2Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213-2683, USA

Received on September 19, 1997; revised on December 29, 1997; accepted on January 2, 1998

The polysaccharide chains of enterobacterial common antigen (ECA) consist of linear trisaccharide repeat units with the structure ->3)-[alpha]-d-Fuc4NAc-(1->4)-[beta]-d-ManNAcA-(1-> 4)-[alpha]-d-GlcNAc-(1->, where Fuc4NAc is 4-acetamido-4,6-dideoxy-d-galactose, ManNAcA is N-acetyl-d- mannosaminuronic acid, and GlcNAc is N-acetyl-d-glucosamine. The major form of ECA (ECAPG) consists of polysaccharide chains that are believed to be covalently linked to diacylglycerol through phosphodiester linkage; the phospholipid moiety functions to anchor molecules in the outer membrane. The ECA trisaccharide repeat unit is assembled as a polyisoprenyl-linked intermediate which has been tentatively identified as Fuc4NAc-ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid III). Subsequent chain-elongation presumably occurs by a block-polymerization mechanism. However, the identity of the polyisoprenoid carrier-lipid has not been established. Accordingly, the current studies were conducted in an effort to structurally characterize the polyisoprenyl lipid-carrier involved in ECA synthesis. Isolation and characterization of the lipid carrier was facilitated by the accumulation of a ManNAcA-GlcNAc-pyrophosphorylpolyisoprenyl lipid (lipid II) in mutants of Salmonella typhimurium defective in the synthesis of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA synthesis. Analyses of lipid II preparations by fast atom bombardment tandem mass spectroscopy (FAB-MS/MS) resulted in the identification of the lipid-carrier as the 55-carbon polyisoprenyl alcohol, undecaprenol. These analyses also resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of this glycolipid revealed its structure to be 1,2-diacyl-sn-glycero-3-pryophosphoryl-GlcNAc-ManNAcA (DGP-disaccharide). An examination of purified ECAPG by phosphorus-31 nuclear magnetic resonance spectroscopy confirmed that the polysaccharide chains are linked to diacylglycerol through phosphodiester linkage. Thus, DGP-disaccharide does not appear to be an intermediate in ECAPG synthesis. Nevertheless, although the available evidence clearly indicate that lipid II is a precursor of DGP-disaccharide, the function of this novel glycolipid is not yet known, and it may be an intermediate in the biosynthesis of a molecule other than ECAPG.

Key words: enterobacterial common antigen/undecaprenol/ phosphoglyceride

Introduction

The enterobacterial common antigen (ECA) is a unique glycolipid located in the outer membrane of all gram-negative enteric bacteria (Mäkelä and Mayer, 1976; Mayer and Schmidt, 1979; Kuhn et al., 1988; Rick and Silver, 1996). The carbohydrate portion of ECA consists of a linear polysaccharide comprised of the amino sugars N-acetyl-d-glucosamine (GlcNAc), N-acetyl-d-mannosaminuronic acid (ManNAcA), and 4-acetamido-4,6-dideoxy-d-Fuc4NAc (Männel and Mayer, 1978; Lugowski et al., 1983). These sugars are linked to one another to form trisaccharide repeat units that have the structure ->3)-[alpha]-d- Fuc4NAc-(1->4)-[beta]-d-ManNAcA-(1->4)-[alpha]-d-GlcNAc-(-> (Lugowski et al., 1983). ECA polysaccharide chains are heterogenous in length, and a given population of molecules is comprised of a homologous series of polymers which differ from one another by a single repeat unit; the apparent Mr of individual chains ranges from approximately 12,000 to 35,000, which corresponds to polysaccharides containing 18-55 repeat units, respectively (Rick et al., 1985). The available information suggests that the polysaccharide chains are covalently linked to diacylglycerol through phosphodiester linkage involving the reducing terminal GlcNAc residue of the first trisaccharide repeat unit (Kuhn et al., 1983); this form of ECA is referred to as ECAPG. Two other minor forms of ECA have also been demonstrated in certain gram-negative enteric bacteria. Thus, the covalent linkage of ECA polysaccharide chains to the core-region of lipopolyasaccharide (ECALPS) has been demonstrated in certain 'rough" organisms that lack O-antigen, but which possess a complete R1, R4, or K-12 LPS core-region (Whang et al., 1972; Schmidt et al., 1976; Kuhn et al., 1988); however, the nature of the linkage of ECA polysaccharide to the LPS core remains to be established. In addition, a water-soluble cyclic form of ECA containing 4-6 repeat units (ECACYC) has been demonstrated in Shigella sonnei phase I (Dell et al., 1984), Yersinia pestis (Vingradov et al., 1994), and Salmonella montevideo (Kuhn et al., 1988; Vingradov et al., 1994). It is important to note that whereas ECAPG is found in all members of the Enterobacteriaceae, the additional occurrence of ECALPS and ECACYC has thus far been demonstrated to occur in relatively few members of this family.

The genetic determinants of ECA in Salmonella and Escherichia coli include genes located in the chromosomal wec gene clusters of these organisms (Lew et al., 1978; Meier and Mayer, 1985; Rick and Silver, 1996). In addition, synthesis of normal amounts of ECA in S.typhimurium is also dependent on genes located in the his-linked wba region (Lew et al., 1986). For example, the rmlA gene encodes TDP-glucose pyrophosphorylase which catalyzes the synthesis of TDP-glucose; TDP-glucose is a precursor of TDP-Fuc4NAc, the donor of Fuc4NAc residues for ECA biosynthesis. Although the synthesis of ECA in [Delta]rmlA mutants should be completely abrogated, these mutants are nevertheless able to synthesize trace amounts of ECA (Lew et al., 1986). The residual synthesis of ECA by these mutants is apparently supported by the synthesis of small amounts of TDP-glucose by an unknown alternative route. Mutants possessing the ECA-trace phenotype are also hypersensitive to the anionic detergent sodium dodecyl sulfate (SDS), and they readily acquire secondary mutations in the wec gene cluster that render the cells both SDS-resistant and ECA-negative (Mäkelä et al., 1976; Rick et al., 1988).

The results of previous studies have established that the ECA trisaccharide repeat unit is assembled as a lipid intermediate according to the following reactions where C55 is believed to be undecaprenol (Rick et al., 1985; Barr and Rick, 1987; Barr et al., 1989):

(1) UDP-GlcNAc + C55-P UMP + GlcNAc-PP-C55 (lipid I)
(2) UDP-ManNAcA + GlcNAc-PP-C55 -> UDP + ManNAcA-GlcNAc-PP-C55 (lipid II)
(3) TDP-Fuc4NAc + ManNAcA-GlcNAc-PP-C55 -> TDP + Fuc4NAc-ManNAcA-GlcNAc-PP-C55 (lipid III)

Subsequent steps presumably involve chain elongation by a block-polymerization mechanism (Whitfield, 1995) followed by transfer of ECA polysaccharide chains from the lipid-carrier to an as yet unidentified diglyceride acceptor to form ECAPG. The availability of only small amounts of these intermediates has precluded obtaining structural data to conclusively identify undecaprenol as the lipid-carrier component. However, an examination of [Delta]rmlA mutants of S.typhimurium revealed that the synthesis of trace amounts of ECA is accompanied by the accumulation of lipid II (Rick et al., 1988). Indeed, the accumulation of lipid II appears to be related in some unknown manner to the hypersensitivity of these mutants to SDS (Rick et al., 1988). The accumulation of lipid II in [Delta]rmlA mutants of S.typhimurium thus afforded the possibility of obtaining sufficient amounts of this intermediate to conclusively characterize the structure of the lipid-carrier moiety. Accordingly, the present work describes the isolation and structural characterization of lipid II from a [Delta]rmlA mutant of S.typhimurium. Analysis of the lipid II by fast atom bombardment tandem mass-spectroscopy (FAB-MS/MS) resulted in the conclusive identification of the lipid-carrier moiety as the 55-carbon polyisoprenoid, undecaprenol. Quite surprisingly, these studies also resulted in the identification of a novel glycolipid which copurified with lipid II. FAB-MS/MS analyses of the glycolipid revealed it to be 1,2-diacyl-sn-glycero-3-pyrophosphoryl-GlcNAc-ManNAcA. The synthesis and possible functions of this lipid are discussed.

Results

Isolation of lipid II

Previous work from this laboratory established that S.typhimurium SH5187 accumulates a lipid-linked intermediate involved in ECA synthesis that has been tentatively identified as ManNAcA-GlcNAc-pyrophosphorylundecaprenol (lipid II) (Rick et al., 1988). However, the limited amounts of lipid II utilized in these studies precluded unequivocal structural characterization of this compound. Accordingly, attempts were made in this study to isolate lipid II in sufficient quantities to enable the conclusive characterization of its structure using fast-atom bombardment tandem mass spectroscopy (FAB-MS/MS). These attempts were facilitated by routinely mixing relatively small quantities of radiolabeled cells obtained from cultures of strain SH5187 incubated with [3H]GlcNAc with large quantities of cells obtained from unlabeled fermentor-grown cells.

DEAE-cellulose chromatography of chloroform-methanol extracts obtained from strain SH5187 following incubation with [3H]GlcNAc revealed a major radioactive peak which contained greater than 90% of the radioactivity following final development of the column with 0.15 M ammonium acetate in chloroform-methanol-water (2:3:1, vol/vol/vol) (Figure 1). Ammonium acetate was removed from the material in pooled fractions 6-8 by Fractogel TSK HW-40 chromatography, and analysis of the material remaining in these fractions by thin-layer chromatography indicated that these fractions contained a single component. Accordingly, a single charred spot with an Rf = 0.3 was detected when thin-layer plates were sprayed with a solution of 3% copper acetate in 8.5% phosphoric acid and then heated at 210°C (Figure 2). In addition, all of the radioactivity in samples migrated with the same chromatographic mobility as did the organic material that was detected by charring (Figure 2). A total of 130-175 nmol of glucosamine was typically detected following total acid hydrolysis of the purified material obtained from 50 l of fermentor grown cells.


Figure 1. DEAE-cellulose chromatography of [3H]GlcNAc-labeled material present in chloroform-methanol extracts of strain SH5187. Cells of strain SH5187 were incubated with [3H]GlcNAc for 30 min during mid-log phase growth, and the cells were subsequently extracted with chloroform/methanol (3:2, v/v). The extracts were applied to the bed of a DEAE-cellulose column (9 cm × 3 cm; acetate form), and the column was washed successively with chloroform:methanol (3:2, v/v) and methanol. The column was then developed with a linear gradient of pyridinium acetate (pH 4.8, 0-4.8 M) which was followed by successive washes with methanol and chloroform/methanol/water (2:3:1, v/v/v). Radiolabeled material was finally eluted from the column following development with 0.15 M ammonium acetate in chloroform/methanol/water (2:3:1, v/v/v); fractions of 7 ml were collected. Additional details are provided in Materials and methods.


Figure 2. Analytical thin-layer chromatography of the lipid II fraction following DEAE-cellulose chromatography. Upper panel, lipid II was extracted from nonradioactive fermentor-grown cells and isolated by DEAE-cellulose chromatography, Fractogel TSK HW-40 chromatography, and thin-layer chromatography as described under Materials and methods. The purity of the lipid II fraction was subsequently determined by thin-layer chromatography using 5 × 20 cm glass-backed silica gel 150A plates (Whatman LK5, 250 µm) using chloroform/methanol/water/concentrated ammonium hydroxide (88:48:10:1, v/v/v/v) as the developing solvent mixture. The plates were then air-dried, and the location of fractionated components was visualized after spraying the plates with 3% copper acetate in 8.5% phosphoric acid followed by heating the plates at 210°C. Lower panel, radiolabeled lipid II was extracted from cells incubated with [3H]GlcNAc, and the radiolabeled material was isolated by DEAE-cellulose chromatography and Fractogel TSK HW-40 chromatography as described under Materials and methods. The radiochemical purity of the material was subsequently determined by thin-layer chromatography using 5 × 20 glass-backed silica gel 150A plates (Whatman LK5, 250 µm) using chloroform/methanol/water/ammonium hydroxide (88:28:10:1, v/v/v/v) as the developing solvent mixture. The plates were air-dried, and the location of radioactivity on the plates was established by scraping silica-gel segments (0.5 cm) from the plates into vials and determining the amount of radioactivity in each segment by liquid scintillation counting.

Negative ion FAB-MS and MS/MS (CAD) spectra

The negative ion FAB-MS spectrum of the purified lipid II showed a prominent molecular ion (M-H)- at 1345 in accordance with that predicted for ManNAcA-GlcNAc-pyrophosphoryl-undecaprenol (lipid II) (Figure 3). The MS/MS (CAD) spectrum for the (M-H)- ion of lipid II revealed several fragment ions in agreement with this assignment (Figure 4). Accordingly, fragment ions characteristic of the diphosphate moiety were evident at m/z 79 (PO3)-, 159 (HP2O6) -, and 177 (H2 P2 O7 )-. A weak fragment ion at m/z 97 (H2PO4)- was also present. In addition, prominent fragment ions at m/z 1275, 1207, 1139, 1071, 1003, 867, 799, 731, and 663 were also observed that are due to random allylic cleavage of the polyisoprenoid chain with the characteristic loss of isoprene units (68 mass units). It should be noted that phosphatidylglycerophosphate was also present in the sample (data not shown), and the molecular ion (M-H)- of this compound at m/z 799 contributed to the prominence of this fragment ion in the FAB/MS spectrum (Figure 3). In addition, the fragment at m/z 935 resulting from allylic cleavage between isoprene units 5 and 6 was obscured by the strong ion fragment at m/z 925 attributable the undecaprenylpyrophosphate moiety (C55H91O7P2)-. The latter fragment ion results from cleavage of the C-O bond involved in the linkage of the lipid II disaccharide to P1 of the pyrophosphate moiety. The structure of the polar region of lipid II was further confirmed by the fragment ions at m/z 579, 499, and 1128. Thus, the fragment ions at m/z 579 and 499 are attributed to ManNAcA-GlcNAc-1-pyrophosphate and ManNAcA-GlcNAc-1-phosphate, respectively, whereas the fragment ion at m/z 1128 results from cleavage of the C-O glycosidic linkage between the anomeric carbon of the nonreducing terminal ManNAcA and GlcNAc.

In addition to the molecular ion for lipid II, prominent fragment ions at m/z 1173 and 1145 were also apparent in the negative FAB-MS spectrum of the purified lipid II sample. The CAD spectra of the fragment ion at m/z 1145 (Figure 5) revealed several characteristic fragment ions that were also present in the CAD spectrum of lipid II (Figure 4). Thus, significant fragment ions at m/z 579 and 499 due to ManNAcA-GlcNAc-1-pyrophosphate and ManNAcA-GlcNAc-1-phosphate, respectively, were evident. However, the CAD spectrum of the m/z 1145 fragment ion included several other unique fragment ions which were not derived from lipid II. These observations supported the conclusion that the lipid II preparation did not consist of a single component. Indeed, the data are consistent with the identification of the m/z 1145 fragment as the molecular ion (M-H)- for the novel glycolipid, 1,2-diacyl-sn-glycero-3-pyrophosphoryl-GlcNAc-ManNAcA (DGP-disaccharide). Accordingly, the m/z fragment ions 725 and 707 (725-H2O) are attributable to diacylglyceropyrophosphate negative ions which result from loss of the GlcNAc-ManNAcA disaccharide due to C-O cleavage between the anomeric carbon of GlcNAc and the oxygen linked to P1 of the pyrophosphate moiety. Indeed, the CAD spectrum for the m/z 725 fragment (Figure 6) supports this conclusion. Thus, the prominent signal for the fragment ion at m/z 159 (HP206)- in the CAD spectrum of the m/z 725 fragment ion, as well as in the CAD spectrum of the m/z 1145 fragment ion, is in agreement with the presence of a pyrophosphoryl-group in the DGP-disaccharide. The fragment ions at m/z 889 (907 - H2O) and 891 (909 - H2O) are consistent with cleavage of the ester-linkages between palmitic (C16:0) and palmitoleic (C 16:1) fatty acyl substituents and the glycerol backbone of DGP-disaccharide, respectively (Figure 5). The corresponding fragment ions at m/z 469 and 471 are also clearly evident in the CAD spectrum of the m/z 725 fragment. The fragment ions at m/z 1019 and 1073 (Figure 5) and m/z 599 and 653 (Figure 6) further indicate the presence of a C16:1 fatty acyl moiety in DGP-disaccharide. As indicated in Figure 5, the m/z 637 fragment ion is attributed to the loss of both C16:0 and C16:1 fatty acyl groups from the glycerol backbone of DGP-disaccharide due to C-O bond cleavage; the corresponding fragment ion (m/z 217) is also clearly evident in the CAD spectrum of the m/z 725 fragment (Figure 6). It should be noted that the data do not allow assignment of the location of fatty acyl groups on the glycerol moiety of the DGP-disaccharide; however, it is assumed that the C16:0 and C16:1 fatty acids are ester-linked to carbons 1 and 2 of the glycerol moiety, respectively, in accordance with the usual location of saturated and unsaturated fatty acyl substituents in bacterial phospholipids (Cronan and Rock, 1987).


Figure 3. Negative ion FAB-MS spectrum of the lipid II fraction. The lipid II fraction obtained following DEAE-cellulose chromatography and Fractogel TSK HW-40 chromatography was analyzed by negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) as described under Materials and methods.


Figure 4. MS/MS (CAD) spectrum of the molecular ion (M-H)- of lipid II. Details are provide under Materials and methods.


Figure 5. MS/MS (CAD) spectrum of the m/z 1145 fragment ion. Details are provided under Materials and methods.


Figure 6. MS/MS (CAD) spectrum of the m/z 725 fragment ion. Details are provided under Materials and methods.

The fragment ion at m/z 1173 (Figure 3) is 28 a.m.u. larger than that of the molecular ion (M-H)- for the DGP-disaccharide structure shown in Figure 5. Thus, although a CAD spectrum for the m/z 1173 fragment ion was not obtained, this fragment is most likely attributable to a homolog of the DGP-disaccharide structure shown in Figure 6 containing either stearate (C18:0) or vaccenic (C18:1) acids as the fatty acyl component in the carbon-1 and carbon-2 positions of the glycerol moiety, respectively. Indeed, the prominent m/z 281 fragment ion in the FAB-MS spectrum (Figure 3) is suggestive of the presence of vaccenic acid. In addition, the presence of a fragment ion at m/z 753 (725 + 28) in the FAB/MS spectrum could be attributed to a similar homolog of the diacylglyceropyrophosphate structure shown in Figure 6. In this regard, it should be noted that structural characterization of the phospholipid aglycone of purified ECAPG has demonstrated the occurrence of palmitic acid as the O-fatty acyl substituent in the 1-position and either palmitic, palmitoleic, cis-vaccenic, or stearic acids as the O-fatty acyl substituent in the 2-position (Kuhn et al., 1983, 1988).

The differential susceptibility of lipid II and DGP-disaccharide to mild-alkaline hydrolysis allowed an estimation of the relative amounts of DGP-disaccharide and lipid II in purified preparations. Lipid II is stable to mild-alkaline hydrolysis. In contrast, a consideration of the structure of DGP-disaccharide predicts that it would be mild-alkaline labile; treatment with mild alkali would be expected to result in the release of ester-linked fatty acyl residues as well as cyclic phosphate formation and subsequent release of radioactive ManNAcA-GlcNAc-1-P as a water-soluble product. Accordingly, treatment of purified [3H]GlcNAc-labeled material (5580 d.p.m.) resulted in the release of [sim]40% of the radioactivity (2420 d.p.m.) as water-soluble material.

Analysis of ECA by phosphorus-31 nuclear magnetic resonance spectroscopy

Although the results of earlier experiments suggested that the polysaccharide chains of ECA are covalently linked to the diacylglycerol moiety through phosphodiester linkage (Kuhn et al., 1983, 1988), the published data only provide rather indirect evidence in support of this conclusion. Moreover, the detection of DGP-disaccharide in an [Delta]rmlA mutant of S.typhimurium in the current study suggested the alternative possibility that the polysaccharide chains of ECAPG might actually be linked to diacylglycerol through pyrophosphate linkage. Accordingly, the assembly of ECAPG molecules might occur by the transfer of either ECA polysaccharide or ECA polysaccharide-1-phosphate from undecaprenylpyrophosphate-linked polysaccharide to the appropriate phosphoglyceride acceptor to yield DGP-polysaccharide by an as yet undetermined mechanism. In this event, the occurrence of DGP-disaccharide in [Delta]rmlA mutants might reflect 'leaky" synthesis of molecules of ECAPG containing a single incomplete repeat unit due to the accumulation of lipid II. In order to examine this possibility, the chemical nature of the phosphate linkage in ECAPG obtained from S.typhimurium was analyzed by phosphorus-31 NMR spectroscopy. The 31P-NMR spectrum of SDS-solubilized ECAPG at pH 5.96 revealed a single sharp resonance 1.3 ppm downfield from that of the triethyl phosphate (TEP) resonance as well as a two weak overlapping resonances at 0.74 and 0.62 ppm (Figure 7). All of these resonances are in the spectral region expected for phosphodiester chemical shifts (Farghali et al., 1964). The presence of more than one resonance can be due to sample heterogeneity or to the presence of more than one type of phosphorylated group in the sample. In fact, although we have consistently detected the resonance at 1.3 ppm, we have also detected inorganic phosphate in some samples that may have arisen as a result of hydrolysis. Except for their phosphodiester character, the exact nature of the resonances shown in Figure 7 cannot be determined from the available information. In addition, the position of these resonances remained unchanged as the pH was increased from pH 5.96 to pH 8.4, which is also characteristic of phosphodiesters (data not shown). No resonances were detected further downfield in the spectral region expected for phosphomonoesters or upfield of TEP in the region expected for the 31P chemical shifts characteristic of pyrophosphates (-17 to -23 ppm). These data provide conclusive evidence that the polysaccharide chains of ECAPG are indeed linked to diacylglycerol through phosphodiester linkage.


Figure 7. 31P-NMR spectrum of SDS-solubilized ECAPG. The inset shows an expanded portion of the spectrum in the region from -1.0 to 3.0 ppm. Details are provided under Materials and methods.

Discussion

A substantial body of data has been obtained in support of the conclusion that the ECA trisaccharide repeat unit is assembled as a polyisoprenoid-linked intermediate (Rick et al., 1985; Barr and Rick, 1987; Barr et al., 1989). Accordingly, the synthesis of ECA polysaccharide chains is initiated by the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to a polyisoprenyl monophosphate acceptor to form a GlcNAc-pyrophosphorylpolyprenol intermediate (lipid I). This conclusion has been supported by observations that both the in vivo synthesis of ECA, as well as the in vitro synthesis of lipid-linked ECA intermediates, are inhibited by the antibiotic, tunicamycin. In addition, biochemical and genetic studies have established that the wecA gene encodes the tunicamycin-sensitive GlcNAc-1-phosphate transferase which catalyzes the synthesis of lipid I (Meier-Dieter et al., 1990, 1992). Numerous other studies have revealed that the synthesis of lipopolysaccharide O-antigen side-chains in a wide variety of Gram-negative enteric bacteria is also wecA-dependent (Rick and Silver, 1996). Recent investigations have demonstrated that E.coli WecA is unable to utilize polyprenyl phosphate acceptors possessing a saturated [alpha]-isoprene unit (Rush et al., 1997). In contrast, the enzyme readily utilizes fully unsaturated polyprenyl phosphate substrates, and maximal activity has been observed with the C55-polyisoprenoid phosphate, undecaprenyl monophosphate. Although undecaprenyl phosphate functions as a lipid-carrier in the synthesis of peptidoglycan (van Heijenoort, 1996) and wecA-independent O-antigens (Whitfield, 1995), conclusive identification of the endogenous polyisoprenoid substrate involved in ECA synthesis has not been accomplished. The results of FAB-MS/MS analyses presented here clearly demonstrate that the C55-polyisoprenoid, undecaprenol, is the polyisoprenoid component of the ManNAcA-GlcNAc-pyrophosphorylpolyprenol ECA intermediate which accumulates in [Delta]rmlA mutants of S.typhimurium. Thus, we conclude that ECA polysaccharide chains are assembled from undecaprenol-linked trisaccharide intermediates, and it seems highly likely that the assembly of wecA-dependent O-antigens also involves undecaprenol-linked intermediates.

The experiments conducted in this investigation also resulted in the unexpected discovery that synthesis and accumulation of lipid II was accompanied by the synthesis and accumulation of a novel class of glycolipid having the general structure, 1,2-diacyl-sn-glycero-3-pyrophosphoryl-GlcNAc-ManNAcA (DGP-disaccharide). Indeed, DGP-disaccharide was found to copurify with lipid II, and it accounted for [sim]40% of the lipid-linked ManAcA-GlcNAc material present in lipid II preparations. The results of FAB-MS/MS analyses indicate that DGP-disaccharide actually consists of a population of molecules which differ only with respect to their fatty-acyl composition. Accordingly, these analyses indicated the occurrence of DGP-disaccharide molecules containing palmitic and palmitoleic acids as well as molecules containing either palmitic and vaccenic acids or stearic and palmitoleic acids. It is also important to note that although is not possible to identify the specific amino sugar moieties of lipid II and DGP-disaccharide by mass spectral analyses alone, the data presented here are consistent with the identification of the disaccharide moiety of these compounds as ManNAcA-GlcNAc.

Essentially nothing is known concerning the mechanism involved in the synthesis of DGP-disaccharide; however, the available data suggest that lipid II is a precursor of this glycolipid. This conclusion is supported by the results of earlier investigations which revealed that the incorporation of radioactivity into lipid II was completely abolished when [Delta]rmlA mutants were incubated with radioactive GlcNAc in the presence of the antibiotic, tunicamycin (Rick et al., 1988). Thus, the synthesis of DGP-disaccharide also appears to be tunicamycin-sensitive since the chromatographic assay used to detect lipid II in these earlier studies does not allow the separation of lipid II from DGP-disaccharide, and DGP-disaccharide accounts for almost half of the ManNAcA-GlcNAc-containing material in the lipid II fraction. Tunicamycin specifically inhibits the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to polyprenyl phosphate acceptors (Takatsuki et al., 1975; Tkacz and Lampen, 1975; Mahoney and Duskin, 1979). Therefore, it appears that tunicamycin does not directly inhibit DGP-disaccharide synthesis but that its synthesis is precluded as a consequence of the inhibition of lipid I synthesis by tunicamycin. Accordingly, the available information support the conclusion that synthesis of DGP-disaccharide is not initiated by the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to phosphatidic acid; rather, it seems likely that this novel glycolipid arises as a result of the transfer ManNAcA-GlcNAc-1-phosphate from lipid II to phosphatidic acid. However, a precursor-product relationship between lipid II and DGP-disaccharides has not yet been demonstrated. It should also be noted that the available data do not preclude a biosynthetic pathway involving the transfer of GlcNAc-1-phosphate from lipid I to phosphatidic acid followed by the incorporation of ManNAcA from the UDP-ManNAcA.

The synthesis and accumulation of lipid II in [Delta]rmlA mutants in Salmonella typhimurium is accompanied by marked hypersensitivity of the mutant strains to sodium dodecyl sulfate (SDS) as well as to other detergents and agents (Mäkelä et al., 1976; Rick et al., 1988). Indeed, spontaneous SDS-resistant derivatives of these mutants emerge with a high frequency, and these derivatives possess suppressor mutations which render the cells unable to synthesize lipid II. Similarly, the inhibition of lipid II synthesis by tunicamycin also renders [Delta]rmlA mutants SDS-resistant. Thus, it is possible that the accumulation of lipid II per se is deleterious to cells and that DGP-disaccharide is synthesized from lipid II in an attempt to alleviate this stress. However, as mentioned previously, a precursor-product relationship between lipid II and this glycolipid has not yet been established. It is also possible that either the accumulation of DGP-disaccharide or the combined accumulation of both lipid II and DGP-disaccharide is responsible for the SDS-hypersensitive phenotype of the [Delta]rmlA mutants of S.typhimurium.

The function of DGP-disaccharide remains to be established. Although the mechanism involved in the formation of the linkage between ECA polysaccharide chains and the phospholipid aglycone of ECAPG has not yet been determined, the synthesis of DGP-disaccharide appears to be unrelated to this process. Accordingly, the 31P-NMR data presented here conclusively demonstrate that the polysaccharide chains of ECAPG are linked to the diglyceride aglycone via phosphodiester linkage. This finding precludes the possibility that the mechanism of ECAPG synthesis results in the linkage of polysaccharide chains to diglyceride via a pyrophosphate bridge. Therefore, the synthesis of DGP-disaccharide does not reflect 'leaky" synthesis of ECAPG molecules containing a single incomplete repeat unit as a consequence of the accumulation of lipid II. Rather, it seems likely that synthesis of ECAPG involves either the transfer of polysaccharide or polysaccharide-1-phosphate from lipid II to phosphatidic acid or diacylglycerol, respectively.

Thus far, no experiments have been conducted to determine if DGP-GlcNAc can be detected in wecB or wecC mutants that accumulate lipid I. Similarly, it is not known if cells are capable of synthesizing DGP-GlcNAc-ManNAcA-Fuc4NAc (DGP-trisaccharide). Accordingly, it is possible that DGP-trisaccharide synthesis and accumulation occurs as a consequence of lipid III accumulation in mutants that are defective in ECA polysaccharide chain elongation. However, significant synthesis and accumulation of DGP-trisaccharide in such mutants might be precluded if the concentration of lipid III in these mutants was low due to the efficient transfer a single trisaccharide repeat unit from lipid III to the appropriate acceptor to form monomeric ECAPG molecules. It is also possible that DGP-disaccharide is not simply a 'dead-end" compound that is synthesized in attempt to alleviate an accumulation of lipid II. Rather, it is possible that lipid III is a precursor of DGP-trisaccharide which in turn is utilized for the synthesis of a molecule other than ECAPG. In this event, lipid II may also be recognized to some extent as an acceptable donor substrate by the relevant enzyme, and the accumulation of DGP-disaccharide may simply reflect the inability of [Delta]rmlA mutants to convert lipid II to lipid III. In this regard, it is interesting to note that the vast majority of pyrophosphate-linked glycoconjugates function as donors of saccharides in biosynthetic reactions. In addition, nothing is known about the mechanism of ECACYC synthesis, and it is tempting to speculate that DGP-trisaccharide might be a biosynthetic intermediate involved in the synthesis of ECACYC. In this event, DGP-trisaccharide might function as a lipid-carrier for the synthesis and assembly of ECACYC in a manner similar to the function of lipid III in the synthesis of linear ECA polysaccharide chains. Although the presence of ECACYC in S.typhimurium has not yet been reported, ECACYC is synthesized by S.montevideo (Kuhn et al., 1988; Vinogradov et al., 1994), and it seems likely that it will also be found in S.typhimurium as well as other Gram-negative enteric organisms. Alternatively, the synthesis of DGP-saccharides may be required for some other previously unrecognized function. For example, the results of studies on the assembly of O-antigens by the block-polymerization mechanism clearly support the conclusion that synthesis of undecaprenylpyrophosphate-linked O-specific repeat units occurs on the cytoplasmic face of the inner membrane and subsequent Wzy-dependent chain-elongation or polymerization of these O-antigens occurs at the external face of the cytoplasmic membrane (McGrath and Osborn, 1991). Although the mechanism involved in the elongation of ECA chains has not been demonstrated, the heteropolysaccharide structure of ECA and the occurrence of putative wzx (Macpherson et al., 1995) and wzy (unpublished observation) homologs in the wec gene cluster suggest that the terminal steps in the assembly of ECA polysaccharide chains also occurs by this mechanism. However, the mechanism involved in the wzx-mediated translocation of either O-antigens or ECA across the cytoplasmic membrane remains to be established. Indeed, although these polymers are assembled as undecaprenyl-linked repeat units and subsequently polymerized to yield undecaprenyl-linked polymers, it has not been unequivocally established that the component repeat units are translocated across the cytoplasmic membrane as undecaprenyl-linked oligosaccharides. Accordingly, it is tempting to speculate that the detection of DGP-linked saccharides as reported here might reflect the occurrence of a previously unrecognized class of phosphoglycerides which function to shuttle saccharide-1-phosphate moieties across the cytoplasmic membrane. Thus, synthesis of DGP-saccharides at the cytoplasmic face of the inner membrane might occur by transfer of a saccharide-1-phosphate from undecaprenylpyrophosphate-saccharide to phosphatidic acid. Subsequent translocation and transfer of saccharide-1-phosphate from DGP-saccharide to undecaprenyl-phosphate localized at the periplasmic face of the membrane would result in the resynthesis of undecaprenylpyrophosphate-linked saccharide which could then serve as a substrate for block polymerization. This reaction would also result in the concomitant generation of phosphatidic acid which could then participate in another cycle following its translocation back to the cytoplasmic face of the membrane.

Materials and methods

Bacterial strains and growth conditions

S.typhimurium strain SH5187 [Delta]rmlA (Mäkelä et al., 1976; Rick et al., 1988) was used for all experiments described in this study. Cultures were grown at 37°C with vigorous aeration in either Proteose Peptone (Difco Laboratories, Detroit, MI) beef extract medium (Rothfield et al., 1964) containing 0.2% glucose (medium A) or Luria-Bertani medium (Maniatis et al., 1982) containing 0.2% glucose (medium B). Large scale growth of the organism for the isolation of lipid II was carried out in a New Brunswick Fermatron Fermentor (Model FM 150). Accordingly, 50 l cultures were grown in medium A at constant pH (pH 7.0) with vigorous aeration at 37°C.

In vivo incorporation of radiolabeled GlcNAc into lipid II

Cultures of strain SH5187 were grown in 200 ml of medium A with vigorous aeration at 37°C until the optical density of the culture at 600 nm reached 0.4. The cells were then concentrated by centrifugation at room temperature, and the cell pellet was resuspended in 25 ml of fresh medium A. The resuspended cells were incubated at room temperature for 5 min without shaking prior to the addition of N-acetyl-d-[1-3H]glucosamine (60 µCi, 11.2 Ci/mmol). The addition of [3H]GlcNAc was accompanied by a shift of the culture to 37°C, and the culture was subsequently incubated with vigorous aeration for 30 min. The culture was next poured over 10 g of crushed ice, and the radiolabeled cells were harvested by centrifugation at 4°C. The cells were then successively washed with cold 0.9% saline (20 ml), 95% ethanol (20 ml) and acetone (20 ml). The extracted cells were dried under vacuum and then stored at -20°C. Radiolabeled cells obtained in this manner were frequently mixed with unlabeled fermentor grown cells to facilitate the purification of lipid II as described below.

Isolation of lipid II

Fermentor grown cells from a 50 l culture were concentrated by filtration when the optical density of the culture at 600 nm reached 1.0; bacterial cells were harvested by centrifugation of the resulting cell suspension at 4°C. The cell pellet was washed with 350 ml cold 0.9% saline and then successively extracted with 350 ml of cold 95% ethanol and 350 ml of cold acetone. The extracted cells were then dried in vacuo and stored at -20°C until processed further. Approximately 9.3 g of extracted and dried cells were typically obtained from 50 l cultures grown in the fermentor.

The isolation of lipid II was initiated by mixing cells with chloroform:methanol (3:2, v/v) to give a ratio of 2 ml solvent mixture/g dried cells followed by constant stirring of the suspension for 60 min at room temperature. The supernatant solution was removed following centrifugation at room temperature, and the particulate fraction was reextracted with the same volume of chloroform/methanol (3:2, v/v). The supernatant solutions were combined and added to a column of DEAE-cellulose (3 cm × 9 cm; acetate form), and the column was subsequently washed with 350 ml of chloroform:methanol (3:2, v/v) followed by 200 ml methanol. The column was then developed with a linear gradient of pyridinium acetate (pH 4.8). More specifically, the mixing chamber contained 100 ml methanol and the reservoir contained 100 ml of a mixture consisting of 20 ml methanol plus 80 ml of 6 M pyridinium acetate (pH 4.8); the pyridinium acetate solution was prepared by titrating glacial acetic acid to pH 4.8 with pyridine and adjusting the volume with water to give a 6 M solution with respect to acetate. The column was then washed with 200 ml of methanol followed by 200 ml of chloroform/methanol/water (2:3:1, v/v/v). Lipid II was eluted from the column during final development of the column with 200 ml of 0.15 M ammonium acetate in chloroform/methanol/water (2:3:1, v/v/v); fractions of 7 ml were collected. The fractions containing radiolabeled lipid II were pooled and taken to dryness in vacuo at room temperature. Ammonium acetate was removed from lipid II preparations by gel-filtration chromatography. Accordingly, the dried material was resuspended in 1.0 ml of chloroform/methanol/water (10:10:3, v/v/v) and applied to a column of Fractogel TSK HW-40 (EM Reagents) (1.5 × 19 cm) equilibrated in methanol. The column was eluted with methanol, and fractions of 1.0 ml were collected. Nonradioactive lipid II was detected in column fractions using thin-layer chromatography. Accordingly, aliquots (50-100 µl) of fractions obtained following DEAE-cellulose and Fractogel TSK HW-40 chromatography were spotted on the preabsorbent strip of 20 × 20 cm glass-backed plates coated with silica gel 150A (Whatman LK5, 250 µm), and the plates were developed at room temperature using chloroform/methanol/water/concentrated ammonium hydroxide (88:48:10:1, v/v/v/v). Following development, the plates were air-dried and subsequently sprayed with a solution of 3% copper acetate in 8.5% phosphoric acid and then heated at 210°C; the lipid II appeared as a single charred spot at Rf = 0.3. Fractions containing lipid II were pooled, concentrated to dryness in vacuo at room temperature, and stored at -20°C under an atmosphere of liquid nitrogen.

[3H]GlcNAc-labeled cells obtained from a 200 ml culture as described above were frequently mixed with nonradioactive fermentor-grown cells in order to facilitate isolation and purification of lipid II. Radioactive lipid II was detected in column fractions by standard liquid scintillation techniques. Purified [3H]GlcNAc-labeled lipid II was also analyzed by thin-layer chromatography as described above. The location of radioactivity on the thin-layer plates was determined by scraping the silica-gel segments (0.5 cm) from the plates into vials and then determining the amount of radioactivity in each segment by standard liquid scintillation counting techniques.

Mild-alkaline treatment of lipid II preparations

Treatment of lipid II with mild-alkali was carried out using the procedure described by Yamamori et al. (Yamamori et al., 1978). Purified [3H]GlcNAc-labeled lipid II (5580 d.p.m.) was incubated with 0.1 N NaOH in 90% ethanol (250 µl) at 75°C for 60 min in a glass tube. The solution was cooled to room temperature and then neutralized by the addition of 1.0 N HCl (25 µl). Chloroform (400 µl) and water (75 µl) were then added with thorough mixing. The resulting organic and aqueous phases were separated by low speed centrifugation at room temperature, and the amount of radioactivityin the aqueous phase was determined.

Isolation and purification of ECA

The isolation and purification of ECA from Salmonella typhimurium LT2 was carried out essentially according to the method of Männel and Mayer (Männel and Mayer, 1978) as described by Heckels and Virji (Heckels and Virji, 1988). The starting material consisted of 20 g of lyophilized cells obtained from fermentor grown cultures. Exceptions to the published procedure were as follows: Following the removal of LPS, the sample was dissolved in 0.5 ml of 85% ethanol and then applied to a 1.5 × 10 cm column of DEAE-cellulose (acetate form, 85% ethanol). The column was successively washed with 85% ethanol (3 ml) and methanol (4 ml) and then eluted with a step-gradient of ammonium acetate in methanol consisting of 0.2 M (1 ml), 0.4 M (1 ml), and 0.6 M ammonium acetate in methanol. The column was finally washed with 1.0 M ammonium acetate in methanol (5 ml). One-milliliter fractions were collected, and the fractions were subsequently evaporated to dryness under a stream of nitrogen. The material in each fraction was then dissolved in 50 µl of water, and fractions containing ECA were identified by an immunoblot procedure using mouse anti-ECA monoclonal antibody mAb898 (Meier-Dieter et al., 1989). Briefly, 3 µl of each sample was spotted on a nitrocellulose filter, and the filter was then dried at 80°C for 4 h. The dried filter was incubated overnight at room temperature in TN/BSA buffer (50 mM Tris-HCl, pH 7.5; 0.9% NaCl; 0.2% sodium azide; bovine serum albumin at a final concentration of 3%). The filter was then rinsed with TN/BSA buffer and subsequently incubated for 2 h at room temperature with a 1:200 dilution of ascites fluid containing mAb898 in TN/BSA buffer. The filter was next rinsed with TN/BSA buffer and incubated for 2 h at room temperature with a 1:1500 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG in TN/BSA buffer. Finally, the filters were developed by incubation in TN/BSA buffer (80 ml) containing 4 µl H2O2 and 3,3[prime]-diaminobenzidine tetrahydrochloride (10 mg).

Phosphorus-31 nuclear magnetic resonance spectroscopy

31P-NMR spectra of ECA were acquired on a Bruker AM500 spectrometer operating at 202 MHz for 31P. Purified ECA (400 nmol phosphate) was dissolved in 0.5 ml CD3OD and the solvent was subsequently removed by evaporation. The sample was then dissolved in 0.5 ml of 6 mM sodium dodecyl sulfate (SDS) at pH 5.96. Spectra were taken with a spectral width of 10 kHz and 2000-10,000 scans. A pulse width of 5.0 µs (45° rotation angle), and a relaxation delay of 3.0 s were employed both with and without broadband decoupling. The temperature was maintained at 27°C using a Bruker variable-temperature unit. Triethyl phosphate (TEP) in D2O was set at 0 ppm as the reference standard. The 31P chemical shift of TEP is the same as that of 85% H3PO4. Spectra were also obtained at pH 6.4 and pH 8.4 following the addition of NaOH to the sample.

Negative ion fast atom bombardment-tandem mass spectrometry analyses

Negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) analyses were carried out as described previously (Kitaoka et al., 1990). FAB/MS spectra and collisionally activated dissociation (CAD) spectra were obtained with a JEOL JMS-HX 100 tandem mass spectrometer (EBE configuration: E, electrostatic field; B, magnetic field) and a JEOL JMS-SX/SX-1028 tandem mass spectrometer (BE/BE configuration). The CAD spectra were obtained by collision with argon gas (sufficient to suppress the precursor ion beam by 25%). Xenon was used to provide the primary beam of atoms ([sim]6K eV). Samples were initially dissolved in chloroform:methanol (3:1, v/v), and 1-2 µl of the resulting solution was mixed with a small amount of triethanolamine prior to its application to the FAB probe. Mass assignments in the mass-analyzed ion kinetic energy (MIKE) spectra obtained with the JEOL JMS-HX 100 instrument in the EB/E configuration were confirmed by the analysis of samples using the JEOL JMS-SX/SX-1028 tandem mass spectrometer in the BE/BE configuration.

Assay procedures

The amount of glucosamine in total acid hydrolysates was determined by the Elson-Morgan reaction as modified by Strominger et al. (Strominger et al., 1959). Total acid hydrolysates were obtained by treating samples with 4 N HCl at 100°C for 10 h in vacuo. Following acid hydrolysis, the samples were reduced to dryness in vacuo over P2O5 and NaOH pellets. Organic and inorganic phosphorous were determined as described by Ames (Ames, 1966).

Genetic nomenclature

The symbols for genes and gene-products in this work are in accordance with the recently formulated Bacterial Polysaccharide Gene Nomenclature (BPGN) scheme (Reeves et al., 1996).

Acknowledgments

This research was supported by United States Public Health Service Grants GM-52882 (P.D.R.), GM-26874 (C.H.), and HL-24525 (C.H.). We dedicate this work to the memory of Henry C. Wu. The views expressed herein are solely those of the authors, and they should not be construed as official or necessarily reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences.

References

Ames,B.N. (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol., 8, 115-118.

Barr,K. and Rick,P.D. (1987) Biosynthesis of enterobacterial common antigen in Escherichia coli. In vitro synthesis of lipid-linked intermediates. J. Biol. Chem., 262, 7142-7150. MEDLINE Abstract

Barr,K., Nunes-Edwards,P. and Rick,P.D. (1989) In vitro synthesis of a lipid-linked trisaccharide involved in synthesis of enterobacterial common antigen. J. Bacteriol., 171, 1326-1332. MEDLINE Abstract

Cronan,J.E.,Jr. and Rock,C.O. (1987) Biosynthesis of membrane lipids. In: Neidhardt,F.C. (ed.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, first ed., Vol. 1, ASM Publications, Washington, DC, pp. 474-497.

Dell,A., Oates,J., Lugowski,C., Romanowska,E. and Lindberg,B. (1984) The enterobacterial common antigen. A cyclic polysaccharide. Carbohydr. Res., 133, 95-104. MEDLINE Abstract

Farghali,H., Rilo,H., Zhang,W., Simplaceanu,V., Gavaler,J.S., Ho,C. and Van Thiel,D.H. (1994) Liver regeneration after partial hepatectomy in the rat. Sequential events monitored by 31P-nuclear magnetic resonance spectroscopy and biochemical studies. Lab. Invest., 70, 418-425. MEDLINE Abstract

Heckels,J.E. and Virji,M. (1988) Separation and purification of surface components. In: Hancock,I.C. and Poxton,I.R., (eds.), Bacterial Cell Surface Techniques. John Wiley & Sons, New York, pp. 67-135.

Kitaoka,M., Nagaki,H., Kinoshita,T., Kurabayashi,M., Koyama,T. and Ogura,K. (1990) Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates. Anal. Biochem., 185, 182-186. MEDLINE Abstract

Kuhn,H.-M., Meier-Dieter,U. and Mayer,H. (1988) ECA, the enterobacterial common antigen. FEMS Microbiol. Rev., 54, 195-222.

Kuhn,H.-M., Neter,E. and Mayer,H. (1983) Modification of the lipid moiety of the enterobacterial common antigen by the 'Pseudomonas" factor. Infect. Immun., 40, 696-700. MEDLINE Abstract

Lew,H., Nikaido,H. and Mäkelä,P.H. (1978) Biosynthesis of uridine diphosphate N-acetyl-mannosaminuronic acid in rff mutants of Salmonella typhimurium. J. Bacteriol., 136, 227-233. MEDLINE Abstract

Lew,H., Mäkelä, P.H., Kuhn,H.-M., Mayer,H. and Nikaido,H. (1986) Biosynthesis of enterobacterial common antigen requires dTDP-glucose pyrophophorylase determined by a Salmonella typhimurium rfb gene and a Salmonella montevideo rfe gene. J. Bacteriol., 168, 715-721. MEDLINE Abstract

Lugowski,C., Romanowska,E., Kenne,L. and Lindberg,B. (1983) Identification of a trisaccharide repeating-unit in the enterobacterial common antigen. Carbohydr. Res., 118, 173-181.

Macpherson,D.F., Manning,P.A. and Morona,R. (1995) Genetic analysis of the rfbX gene of Shigella flexneri. Gene, 155, 9-17. MEDLINE Abstract

Mahoney,W.C., and Duskin,D. (1979) Biological activities of two major components of tunicamycin. J. Biol. Chem., 254, 6572-6576. MEDLINE Abstract

Mäkelä,P.H. and Mayer,H. (1976) Enterobacterial common antigen. Bacteriol. Rev., 40, 591-632. MEDLINE Abstract

Mäkelä,P.H., Schmidt,G., Mayer,H., Nikaido,H., Whang,H.Y. and Neter,E. (1976) Enterobacterial common antigen in rfb deletion mutants of Salmonella typhimurium. J. Bacteriol., 127, 1141-1149. MEDLINE Abstract

Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Männel,D. and Mayer,H. (1978) Isolation and chemical characterization of the entoerbacterial common antigen. Eur. J. Biochem., 86, 361-370. MEDLINE Abstract

Mayer,H. and Schmidt,G. (1979) Chemistry and biology of the enterobacterial common antigen (ECA). Curr. Top. Microbiol. Immunol., 85, 99-153. MEDLINE Abstract

McGrath,B.C. and Osborn,M.J. (1991) Localization of the terminal steps of O-antigen synthesis in Salmonella typhimurium. J. Bacteriol., 173, 649-654. MEDLINE Abstract

Meier,U. and Mayer,H. (1985) Genetic location of genes encoding enterobacterial common antigen. J. Bacteriol., 163, 756-762. MEDLINE Abstract

Meier-Dieter,U., Acker,G. and Mayer,H. (1989) Detection of enterobacterial common antigen on bacterial cell surfaces by colony-immunoblotting: effect of its linkage to lipopolysaccharide. FEMS Microbiol. Lett., 59, 215-220.

Meier-Dieter,U., Starman,R., Barr,K., Mayer,H. and Rick,P.D. (1990) Biosynthesis of enterobacterial common antigen in Escherichia coli. Biochemical characterization of Tn10 insertion mutants defective in enterobacterial common antigen synthesis. J. Biol. Chem., 265, 13490-13497. MEDLINE Abstract

Meier-Dieter,U., Barr,K., Starman,R., Hatch,L. and Rick,P.D. (1992) Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster. J. Biol. Chem., 267, 746-753. MEDLINE Abstract

Reeves,P.R., Hobbs,M., Valvano,M.A., Skurnik,M., Whitfield,C., Coplin,D., Kido,N., Klena,J., Maskell,D., Raetz,C. and Rick,P.D. (1996) Bacterial polysaccharide synthesis and gene nomenclature. Trends Microbiol., 4, 495-503. MEDLINE Abstract

Rick,P.D. and Silver,R.P. (1996) Enterobacterial common antigen and capsular polysaccharides. In: Neidhardt,F.C. (ed.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, second edition, Vol. 1. ASM Publications, Washington, DC, pp. 104-122.

Rick,P.D., Mayer,H., Neumeyer,B.A., Wolski,S. and Bitter-Suermann,D. (1985) Biosynthesis of enterobacterial common antigen. J. Bacteriol., 162, 494-503. MEDLINE Abstract

Rick,P.D., Wolski,S., Barr,K., Ward,S. and Ramsay-Sharer,L. (1988) Accumulation of a lipid-linked intermediate involved in enterobacterial common antigen synthesis in Salmonella typhimurium mutants lacking dTDP-glucose pyrophosphorylase. J. Bacteriol., 170, 4008-4014. MEDLINE Abstract

Rothfield,L., Osborn,M.J. and Horecker,B.L. (1964) Biosynthesis of bacterial lipopolysaccharide. II. Incorporation of glucose and galactose catalyzed by particulate and soluble enzymes in Salmonella. J. Biol. Chem., 239, 2788-2795.

Rush,J.S., Rick,P.D. and Waechter,C.J. (1997) Polyisoprenyl phosphate specificity of UDP-GlcNAc:undecaprenyl phosphate N-acetylglucosaminyl 1-P transferase from E.coli. Glycobiology, 7, 315-322. MEDLINE Abstract

Schmidt,G.,Männel,D., Mayer,H., Whang,H.Y. and Neter,E. (1976) The role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen. J. Bacteriol., 126, 579-586. MEDLINE Abstract

Strominger,J.L., Park,J.T. and Thompson,R.E. (1959) Composition of the cell wall ofStaphylococcus aureus: its relation to the mechanism of action of penicillin. J. Biol. Chem., 234, 3263-3268.

Takatsuki,A., Kohno,K. and Tamura,G. (1975) Inhibition of biosynthesis of polyisoprenol sugars in chick embryo microsomes by tunicamycin. Agr. Biol. Chem., 39, 2089-2091.

Tkacz,J.S. and Lampen,O. (1975) Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem. Biophys. Res. Commun., 65, 248-257. MEDLINE Abstract

van Heijenoort,J. (1996) Murein synthesis. In: Neidhardt,F.C. (ed.), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, second edition, Vol. 1. ASM Publications, Washington, DC, pp. 1025-1034.

Vinogradov,E.V., Knirel,Y.A., Thomas-Oates,J.E., Shaskov,A.S. and L'vov,V.L. (1994) The structure of the cyclic enterobacterial common antigen (ECA) from Yersinia pestis. Carbohydr. Res., 258, 223-232. MEDLINE Abstract

Whang,H.Y., Mayer,H., Schmidt,G. and Neter,E. (1972) Immunogenicity of the common enterobacterial antigen produced by smooth and rough strains. Infect. Immun., 6, 533-539. MEDLINE Abstract

Whitfield,C. (1995) Biosynthesis of lipopolysaccharide O-antigens. Trends Microbiol., 3, 178-185. MEDLINE Abstract

Yamamori,S., Mirazumi,N., Araki,Y. and Ito,E. (1978) Formation and function of N-acetyl-glucosamine-linked phosphoryl- and pyrophosphorylundecaprenols in membranes from Bacillus cereus. J. Biol. Chem., 253, 6516-6522. MEDLINE Abstract

3To whom correspondence should be addressed at: Department of Microbiology and Immunology, The Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799.

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 18 May 1998
Copyright©Oxford University Press, 1998.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Appl. Environ. Microbiol.Home page
T. Bottiger, T. Schneider, B. Martinez, H.-G. Sahl, and I. Wiedemann
Influence of Ca2+ Ions on the Activity of Lantibiotics Containing a Mersacidin-Like Lipid II Binding Motif
Appl. Envir. Microbiol., July 1, 2009; 75(13): 4427 - 4434.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. Ruiz
Bioinformatics identification of MurJ (MviN) as the peptidoglycan lipid II flippase in Escherichia coli
PNAS, October 7, 2008; 105(40): 15553 - 15557.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
B. Martinez, T. Bottiger, T. Schneider, A. Rodriguez, H.-G. Sahl, and I. Wiedemann
Specific Interaction of the Unmodified Bacteriocin Lactococcin 972 with the Cell Wall Precursor Lipid II
Appl. Envir. Microbiol., August 1, 2008; 74(15): 4666 - 4670.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Touze, D. Blanot, and D. Mengin-Lecreulx
Substrate Specificity and Membrane Topology of Escherichia coli PgpB, an Undecaprenyl Pyrophosphate Phosphatase
J. Biol. Chem., June 13, 2008; 283(24): 16573 - 16583.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
J. Parisot, S. Carey, E. Breukink, W. C. Chan, A. Narbad, and B. Bonev
Molecular Mechanism of Target Recognition by Subtilin, a Class I Lanthionine Antibiotic
Antimicrob. Agents Chemother., February 1, 2008; 52(2): 612 - 618.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Yan, Z. Guan, and C. R. H. Raetz
An Undecaprenyl Phosphate-Aminoarabinose Flippase Required for Polymyxin Resistance in Escherichia coli
J. Biol. Chem., December 7, 2007; 282(49): 36077 - 36089.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
J. Kajimura, A. Rahman, J. Hsu, M. R. Evans, K. H. Gardner, and P. D. Rick
O Acetylation of the Enterobacterial Common Antigen Polysaccharide Is Catalyzed by the Product of the yiaH Gene of Escherichia coli K-12
J. Bacteriol., November 1, 2006; 188(21): 7542 - 7550.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. El Ghachi, A. Bouhss, H. Barreteau, T. Touze, G. Auger, D. Blanot, and D. Mengin-Lecreulx
Colicin M Exerts Its Bacteriolytic Effect via Enzymatic Degradation of Undecaprenyl Phosphate-linked Peptidoglycan Precursors
J. Biol. Chem., August 11, 2006; 281(32): 22761 - 22772.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
M.-N. Hung, E. Rangarajan, C. Munger, G. Nadeau, T. Sulea, and A. Matte
Crystal Structure of TDP-Fucosamine Acetyltransferase (WecD) from Escherichia coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis.
J. Bacteriol., August 1, 2006; 188(15): 5606 - 5617.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
R. R. Bonelli, T. Schneider, H.-G. Sahl, and I. Wiedemann
Insights into In Vivo Activities of Lantibiotics from Gallidermin and Epidermin Mode-of-Action Studies.
Antimicrob. Agents Chemother., April 1, 2006; 50(4): 1449 - 1457.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
I. Wiedemann, T. Bottiger, R. R. Bonelli, T. Schneider, H.-G. Sahl, and B. Martinez
Lipid II-Based Antimicrobial Activity of the Lantibiotic Plantaricin C
Appl. Envir. Microbiol., April 1, 2006; 72(4): 2809 - 2814.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
R. T. Cartee, W. T. Forsee, M. H. Bender, K. D. Ambrose, and J. Yother
CpsE from Type 2 Streptococcus pneumoniae Catalyzes the Reversible Addition of Glucose-1-Phosphate to a Polyprenyl Phosphate Acceptor, Initiating Type 2 Capsule Repeat Unit Formation
J. Bacteriol., November 1, 2005; 187(21): 7425 - 7433.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
J. Kajimura, A. Rahman, and P. D. Rick
Assembly of Cyclic Enterobacterial Common Antigen in Escherichia coli K-12
J. Bacteriol., October 15, 2005; 187(20): 6917 - 6927.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
Y.-L. Tzeng, A. K. Datta, C. A. Strole, M. A. Lobritz, R. W. Carlson, and D. S. Stephens
Translocation and Surface Expression of Lipidated Serogroup B Capsular Polysaccharide in Neisseria meningitidis
Infect. Immun., March 1, 2005; 73(3): 1491 - 1505.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. D. Rick, K. Barr, K. Sankaran, J. Kajimura, J. S. Rush, and C. J. Waechter
Evidence That the wzxE Gene of Escherichia coli K-12 Encodes a Protein Involved in the Transbilayer Movement of a Trisaccharide-Lipid Intermediate in the Assembly of Enterobacterial Common Antigen
J. Biol. Chem., May 2, 2003; 278(19): 16534 - 16542.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
P. J. A. Erbel, K. Barr, N. Gao, G. J. Gerwig, P. D. Rick, and K. H. Gardner
Identification and Biosynthesis of Cyclic Enterobacterial Common Antigen in Escherichia coli
J. Bacteriol., March 15, 2003; 185(6): 1995 - 2004.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
A. Rahman, K. Barr, and P. D. Rick
Identification of the Structural Gene for the TDP-Fuc4NAc:Lipid II Fuc4NAc Transferase Involved in Synthesis of Enterobacterial Common Antigen in Escherichia coli K-12
J. Bacteriol., November 15, 2001; 183(22): 6509 - 6516.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. S. Trent, A. A. Ribeiro, W. T. Doerrler, S. Lin, R. J. Cotter, and C. R. H. Raetz
Accumulation of a Polyisoprene-linked Amino Sugar in Polymyxin-resistant Salmonella typhimurium and Escherichia coli. STRUCTURAL CHARACTERIZATION AND TRANSFER TO LIPID A IN THE PERIPLASM
J. Biol. Chem., November 9, 2001; 276(46): 43132 - 43144.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
K. Barr, J. Klena, and P. D. Rick
The Modality of Enterobacterial Common Antigen Polysaccharide Chain Lengths Is Regulated by o349 of the wec Gene Cluster of Escherichia coli K-12
J. Bacteriol., October 15, 1999; 181(20): 6564 - 6568.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (23)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Rick, P. D.
Right arrow Articles by Ho, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rick, P. D.
Right arrow Articles by Ho, C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?