Division of Medical and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, D-23845 Borstel, Germany, Department of Chemistry, Carlsberg Laboratory, DK-2500 Valby, Denmark, Department of Mass Spectrometry, Bijvoet Center for Biomolecular Research, Utrecht University,NL-3584 CA Utrecht, The Netherlands
For the first time, the tetrasaccharide Kdo a2→5 Kdo a2→5 (Kdo a2→4)Kdo (Kdo is 3-deoxy-Dmanno-oct-2-ulopyranosonic acid) has been identified in a bacterial lipopolysaccharide (LPS), i.e. in the core region of LPS from Acinetobacter baumannii NCTC 10303. The LPS was analyzed using compositional analysis, mass spectrometry, and NMR spectroscopy. The disaccharide DGlcpN b1→6 DGlcpN, phosphorylated at O-1 and O-4', was identified as the carbohydrate backbone of the lipid A. The Kdo tetrasaccharide is attached to O-6' of this disaccharide and is further substituted by short L-rhamnoglycans of varying length and by the disaccharide DGlcpNAc a1→4 DGlcpNA (GlcpNA, 2-amino-2-deoxy-glucopyranosuronic acid). The core region is not substituted by phosphate residues and represents a novel core type of bacterial LPS. The complete carbohydrate backbone of the LPS is shown in Structure I as follows: [see formula in text] where Rha is rhamnose. Except were indicated, monosaccharides possess the D-configuration. Sugars marked with an asterisk are present in non-stoichiometric amounts.
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12
0,6,2 lR3HOLau ? 44.1 69.1 36.3 21.0-33.0 21.0-33.0 21.0-33.0 21.0-33.0 21.0-33.0 21.0-33.0 21.0-33.0 13.9
0,6,4 P
0,6,6,4 aXKdop ? ? 34.8 65.6 67.4 72.4 71.8 63.6
0,6,6,5,4,4 aDGlcpN 95.1 54.6 69.9 69.9 72.9 60.4
0,6,6,5,4 aDGlcpNA 91.5 54.0 71.2 75.2 72.3 ?
0,6,6,5,5,8,3,3 aLRhap 102.7 70.6 70.5 72.4 69.5 ?
0,6,6,5,5,8,3 aLRhap 102.6 70.4 78.8 71.7 69.6 ?
0,6,6,5,5,8 aLRhap 100.8 70.2 79.1 71.1 69.2 ?
0,6,6,5,5 aXKdop ? ? 36.7 66.2 67.7 75.6 72.8 68.1
0,6,6,5 aXKdop ? ? 34.7 68.2 69.7 74.2 71.1 63.1
0,6,6 aXKdop ? ? 34.7 73.5 67.4 73.3 69.8 64.3
0,6 bDGlcpN 101.4 55.4 74.5-74.6 74.5-74.6 74.5-74.6 63.6
0 aDGlcpN 91.3 55.0 70.1 69.8 73.0 68.3
P
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12
0,6,2 lR3HOLau - 2.481 4.022 1.515 1.280 1.280 1.280 1.280 1.280 1.280 1.280 0.860
0,6,4 P
0,6,6,4 aXKdop - - 1.744-1.998 4.031 3.989 3.557 3.926 3.716-4.003
0,6,6,5,4,4 aDGlcpN 5.745 3.254 3.848 3.423 3.807 ?
0,6,6,5,4 aDGlcpNA 5.280 3.469 4.108 3.915 4.153 -
0,6,6,5,5,8,3,3 aLRhap 5.046 4.070 3.844 3.453 3.838 1.292
0,6,6,5,5,8,3 aLRhap 5.032 4.159 3.911 3.560 3.887 1.300
0,6,6,5,5,8 aLRhap 4.841 4.090 3.850 3.552 3.748 1.300
0,6,6,5,5 aXKdop - - 1.869-2.299 4.302 3.942 4.240 4.206 3.492-4.495
0,6,6,5 aXKdop - - 2.056-2.432 4.463 4.195 4.378 4.000 3.990-3.990
0,6,6 aXKdop - - 1.974-2.081 3.960 4.297 3.503 3.684 3.547-3.857
0,6 bDGlcpN 4.691 3.766 3.720 3.752 3.680 3.381-3.666
0 aDGlcpN 5.646 3.348 3.910 3.629 4.050 3.819-4.097
P
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6 C7/H7 C8/H8 C9/H9 C10/H10 C11/H11 C12/H12
0,6,2 lR3HOLau 44.1/2.481 69.1/4.022 36.3/1.515 21.0-33.0/1.280 21.0-33.0/1.280 21.0-33.0/1.280 21.0-33.0/1.280 21.0-33.0/1.280 21.0-33.0/1.280 21.0-33.0/1.280 13.9/0.860
0,6,4 P
0,6,6,4 aXKdop 34.8/1.744-1.998 65.6/4.031 67.4/3.989 72.4/3.557 71.8/3.926 63.6/3.716-4.003
0,6,6,5,4,4 aDGlcpN 95.1/5.745 54.6/3.254 69.9/3.848 69.9/3.423 72.9/3.807 60.4/?
0,6,6,5,4 aDGlcpNA 91.5/5.280 54.0/3.469 71.2/4.108 75.2/3.915 72.3/4.153
0,6,6,5,5,8,3,3 aLRhap 102.7/5.046 70.6/4.070 70.5/3.844 72.4/3.453 69.5/3.838 ?/1.292
0,6,6,5,5,8,3 aLRhap 102.6/5.032 70.4/4.159 78.8/3.911 71.7/3.560 69.6/3.887 ?/1.300
0,6,6,5,5,8 aLRhap 100.8/4.841 70.2/4.090 79.1/3.850 71.1/3.552 69.2/3.748 ?/1.300
0,6,6,5,5 aXKdop 36.7/1.869-2.299 66.2/4.302 67.7/3.942 75.6/4.240 72.8/4.206 68.1/3.492-4.495
0,6,6,5 aXKdop 34.7/2.056-2.432 68.2/4.463 69.7/4.195 74.2/4.378 71.1/4.000 63.1/3.990-3.990
0,6,6 aXKdop 34.7/1.974-2.081 73.5/3.960 67.4/4.297 73.3/3.503 69.8/3.684 64.3/3.547-3.857
0,6 bDGlcpN 101.4/4.691 55.4/3.766 74.5-74.6/3.720 74.5-74.6/3.752 74.5-74.6/3.680 63.6/3.381-3.666
0 aDGlcpN 91.3/5.646 55.0/3.348 70.1/3.910 69.8/3.629 73.0/4.050 68.3/3.819-4.097
P
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