The lipopolysaccharide (LPS) O-antigen ofGne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membranes of gram-negative bacteria. The LPS molecule contains three structural parts: (i) the O-antigen, a polysaccharide region that protrudes into its surroundings; (ii) the core, an oligosaccharide often rich in negatively charged groups; and (iii) the lipid A, a polar glycolipid in which a disaccharide backbone is replaced with six or seven saturated fatty acids (33). It is common for the genes required for synthesis of the different LPS parts to be clustered in separated loci. In Yersinia, the O-antigen gene cluster (hereafter referred to as the wb cluster) lies between the hemH and gsk genes in the chromosome (37). This location is different from those in Escherichia coli and Salmonella enterica, where the wb cluster is closely linked to the gnd locus, which lies upstream of the his operon (6, 35).The biosynthesis of heteropolymeric O-antigens whose O-units are composed of different sugar residues starts in the cytoplasm by the activation of sugar 1-phosphates in a reaction with one of the nucleoside triphosphates, followed by different biosynthetic pathways to give rise to individual nucleoside diphosphate (NDP)-activated sugar precursors. The assembly of the O-units takes place on the cytoplasmic face of the inner membrane and involves an initiation process that transfers the first sugar residue from the NDP sugar precursor onto a lipid carrier, undecaprenylphosphate, followed by sequential transfer of the other sugar residues from the respective NDP sugar precursors by specific glycosyltransferases. The completed O-units still assembled on the undecaprenylphosphate are translocated by the O-unit flippase, Wzx, across the inner membrane to the periplasmic face. There, the O-units are polymerized by the O-antigen polymerase, Wzy, into an O-antigen that is ligated to the lipid A core structure by the O-antigen ligase encoded by the waaL gene of the core gene cluster (for a review, see reference 49).The O-antigen polysaccharide of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repe...

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The lipopolysaccharide (LPS) of strain 8081-c-R2, a spontaneous R-mutant of Yersinia enterocolitica serotype O:8, was isolated using extraction with phenol/chloroform/ light petroleum. Its compositional analysis indicated the presence of d-GlcN, d-Glc, l-glycero-d-manno-and dglycero-d-manno-heptose, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and phosphate. From deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide, three oligosaccharides (1±3) were isolated using high-performance anion-exchange chromatography, the structures of which were determined by compositional analysis and one-and two-dimensional NMR spectroscopy as in which all sugars are pyranoses, and R and R H represent b-d-Glc (in 1 and 2) and b-d-GlcN (in 1 only), respectively. d-a-d-Hep is d-glycero-a-d-manno-heptose, l-a-d-Hep isl-glycero-a-d-manno-heptose, Kdo is 3-deoxy-d-mannooct-2-ulosonic acid, and P is phosphate. The liberated lipid A was analyzed by compositional analyses and MALDI-TOF MS. Its b-d-GlcN4P-(136)-a-d-GlcN-13P backbone is mainly tetra-acylated with two amide-and one ester-linked (at O3 of the reducing GlcN) (R)-3-hydroxytetradecanoic acid residues, and one tetradecanoic acid that is attached to the 3-OH group of the amide-linked (R)-3-hydroxytetradecanoic acid of the nonreducing GlcN. Additionally, small amounts of tri-and hexa-acylated lipid A species occur.

L-Glycero-D-manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria. In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L-glycero-D-manno-heptopyranose to Re-LPS and Rd 2 -LPS, respectively. It had been proposed that both reactions involve ADPL-glycero-D-manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated. In the present study, ADPL-glycero-D-manno-heptose was isolated from a heptosyltransferasedeficient E. coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL-glycero-β-D-manno-heptopyranose. This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro.

Die Lipopolysaccharide (LPS) der Gram-negativen Bakterien sind essentielle Bestandteile der äuûeren Membran. Der enylgruppen anfällig für Zersetzungsreaktionen unter Bildung von metallischem Antimon. Der gleiche Trend zeigt sich bei den Molekülen P 4 , As 4 und Sb 4 . Es ist zu prüfen, ob durch einen entsprechenden sterischen Schutz auch die Isolierung von Komplexen mit dem cyclo-Sb 6 -Liganden ermöglicht werden kann und ob Bismut eine ähnliche Chemie aufweist. Experimentelles1 ± 3: Alle Arbeiten wurden unter strengem Luftausschluss in einer Argonatmosphäre oder im Vakuum durchgeführt. cyclo-tBu 4 Sb 4 (1.88 g, 2.63 mmol) und [Cp'''(CO) 3 MoCH 3 ] (1 g, 2.33 mmol) wurden in 60 mL Dekalin ca. 3 h unter Rückfluss erhitzt. Das Lösungsmittel wurde im Vakuum entfernt und der Rückstand mit 3 Â 100 mL Hexan extrahiert. Die grüngelben Extrakte wurden vereinigt, mit 8 g Al 2 O 3 versetzt und das Lösungsmittel wurde unter vermindertem Druck entfernt. Der Rückstand wurde auf eine Chromatographiesäule (3 Â 20 cm neutrales Al 2 O 3 , Korngröûe 0.063 ± 0.200 mm, Aktivität II nach Brockmann) aufgetragen. Mit Hexan/Toluol (99/1) wurde zunächst eine smaragdgrüne Fraktion eluiert. Nach Entfernen des Lösungsmittels im Vakuum und Umkristallisieren des Rückstands aus Hexan bei À 30 8C wurden 265 mg eines mikrokristallinen, grünen Festoffes bestehend aus 1 und 2 isoliert. Durch Lösen in Toluol und langsames Abkondensieren des Lösungsmittels im Vakuum wurde ein Gemisch eines mikrokristallinen Pulvers mit gröûeren Kristallen erhalten, die kristallographisch untersucht wurden und bei denen es sich um 2 handelte. 1 2: Schmp. 331 ± 333 8C; Elementaranalyse (%): ber. für C 34 H 58 Mo 2 Sb 5 /C 31 H 52 Mo 2 Sb 5 : C 32.22/30.39, H 4.61/4.29; gef.: C 32.18, H 4.63; 1 H-NMR (250 MHz, C 6 D 6 , 258C): d 0.91 (br. s), 1.35 (br. s), 1.38 (br. s), 3.39 (br. s), 3.58 (br. s), 3.62 (br. s), 3.77 (br. s), 29.04 (br. s); MS (EI, 70 eV): m/z (%): 1266 (100) [1, M ], 1224 (25) [2, M ], 1195 (20) [1, M À C 5 H 11 ], 1153 (10) [2, M À C 5 H 11 ], 1122 (20), 57 (80) [tBu ]. Mit Hexan/ Toluol (9/1) wurde eine gelbbraune Fraktiuon eluiert, aus der nach Entfernen des Lösungsmittels 3 als dunkelbrauner Feststoff isoliert wurde. Kristalle wurden durch Abkühlen einer Hexanlösung auf À 30 8C erhalten. Ausbeute 205 mg (22.8 %). 3: Schmp. 185 ± 190 8C; Elementaranalyse (%): ber. für C 38 H 58 Mo 2 O 4 : C 59.cm À1 (CO); MS (EI, 70 eV): m/z (%): 770 (80) [M ], 742 (60) [M À CO], 714 (40) [M À 2 CO], 686 (30) [M À 3 CO], 650 (43), 57 (100) [tBu ].