Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1065
Colanic acid (CA) or M-antigen is an exopolysaccharide produced by many enterobacteria, including the majority of Escherichia coli strains. Unlike other capsular polysaccharides which have a close association with the bacterial surface, CA forms a loosely associated saccharide mesh that coats the bacteria, often within biofilms. Herein we show that a highly mucoid strain of E. coli K-12 ligates CA repeats to a significant proportion of lipopolysaccharide (LPS) core acceptor molecules, forming the novel LPS glycoform we call MLPS. MLPS biosynthesis is dependent upon i) CA induction, ii) LPS core biosynthesis, and iii) the O-antigen ligase WaaL. Compositional analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy of a purified MLPS sample confirmed the presence of a CA repeat unit identical in carbohydrate sequence, but differing at multiple positions in anomeric configuration and linkage, from published structures of extracellular CA. The attachment point was identified as O-7 of the L-glycero-D-manno-heptose of the outer LPS core, the same position used for O-antigen ligation. When O-antigen biosynthesis was restored in the K-12 background and grown under conditions meeting the above specifications, only MLPS was observed, suggesting E. coli can reversibly change its proximal covalently linked cell surface polysaccharide coat from O-antigen to CA in response to certain environmental stimuli. The identification of MLPS has implications for potential underlying mechanisms coordinating the synthesis of various surface polysaccharides
13C NMR, 1H NMR, methylation, ESI-MS, acid hydrolysis, composition analysis, NMR-1D, serological methods, genetic methods
13C NMR data:
Linkage Residue C1 C2 C3 C4 C5 C6 C7
5,3,3,3,2,6,7,3,4,3,3,4,6 xRPyr 175.96 101.19 26.23
5,3,3,3,2,6,7,3,4,3,3,4 aDGalp 100.80 68.95 68.95 72.06 63.68 66.21
5,3,3,3,2,6,7,3,4,3,3 bDGlcpA 104.98 73.92 76.95 78.65 76.14 174.83
5,3,3,3,2,6,7,3,4,3,2 Ac 174.29 21.39
5,3,3,3,2,6,7,3,4,3 aDGalp 99.36 70.98 77.58 67.11 ? ?
5,3,3,3,2,6,7,3,4 aLFucp 101.55 ? ? 72.70 68.22 16.43
5,3,3,3,2,6,7,3,2 %Ac 174.29 21.98
5,3,3,3,2,6,7,3,3 %Ac 174.29 21.39
5,3,3,3,2,6,7,3 aLFucp 97.95 71.79 67.60 81.25 68.51 16.23
5,3,3,3,2,6,7 bDGlcp 103.27 74.27 83.02 69.19 77.01 61.73
5,3,3,3,2,6 aXLDmanHepp 100.36 70.96 71.99 67.16 72.49 68.27 73.07
5,3,3,3,2 aDGlcp 96.97 72.50 74.36 70.27 71.07 65.71
5,3,3,3 aDGlcp 97.93 76.60 72.05 70.28 72.45 61.02
5,3,3,6 aDGalp 99.24 69.66 70.56 70.25 ? ?
5,3,3 aDGlcp 102.56 71.35 81.42 ? ? 65.99
5,3,4 P
5,3,7 aXLDmanHepp 100.91 ? ? ? ? ? ?
5,3 aXLDmanHepp 103.41 70.93 80.77 69.63 ? ? ?
5,4,0 %xXEtN 63.48 41.09
5,4 P
5 aXLDmanHepp 101.51 ? ? ? ? ? ?
aXKdo?
1H NMR data:
Linkage Residue H1 H2 H3 H4 H5 H6 H7 H8
5,3,3,3,2,6,7,3,4,3,3,4,6 xRPyr - - 1.466
5,3,3,3,2,6,7,3,4,3,3,4 aDGalp 5.454 3.914 3.914 4.210 3.751 3.874-4.005
5,3,3,3,2,6,7,3,4,3,3 bDGlcpA 4.680 3.395 3.764 3.796 3.921 -
5,3,3,3,2,6,7,3,4,3,2 Ac - 2.133
5,3,3,3,2,6,7,3,4,3 aDGalp 5.352 5.083 4.319 4.240 ? ?
5,3,3,3,2,6,7,3,4 aLFucp 4.979 3.935 4.033 3.991 4.451 1.210
5,3,3,3,2,6,7,3,2 %Ac - 2.210
5,3,3,3,2,6,7,3,3 %Ac - 2.159
5,3,3,3,2,6,7,3 aLFucp 5.388 4.910 4.199 3.918 4.409 1.264
5,3,3,3,2,6,7 bDGlcp 4.493 3.536 3.655 3.482 3.472 3.684-3.920
5,3,3,3,2,6 aXLDmanHepp 4.893 3.973 3.818 3.859 3.607 4.221 3.856-4.057
5,3,3,3,2 aDGlcp 5.171 3.610 3.746 3.525 4.145 3.659-3.950
5,3,3,3 aDGlcp 5.462 3.678 3.850 3.541 4.068 3.819-3.906
5,3,3,6 aDGalp 5.002 3.835 3.899 4.043 ? ?
5,3,3 aDGlcp 5.197 3.663 3.941 3.94 4.033 3.764-4.084
5,3,4 P
5,3,7 aXLDmanHepp 4.927 ? ? ? ? ? ?
5,3 aXLDmanHepp 5.130 4.410 4.111 4.438 ? ? ?
5,4,0 %xXEtN 4.218 3.298
5,4 P
5 aXLDmanHepp 5.282 ? ? ? ? ? ?
aXKdo? - - 1.907-2.270 4.150 ? ? ? ?
1H/13C HSQC data:
Linkage Residue C1/H1 C2/H2 C3/H3 C4/H4 C5/H5 C6/H6 C7/H7 C8/H8
5,3,3,3,2,6,7,3,4,3,3,4,6 xRPyr 26.23/1.466
5,3,3,3,2,6,7,3,4,3,3,4 aDGalp 100.80/5.454 68.95/3.914 68.95/3.914 72.06/4.210 63.68/3.751 66.21/3.874-4.005
5,3,3,3,2,6,7,3,4,3,3 bDGlcpA 104.98/4.680 73.92/3.395 76.95/3.764 78.65/3.796 76.14/3.921
5,3,3,3,2,6,7,3,4,3,2 Ac 21.39/2.133
5,3,3,3,2,6,7,3,4,3 aDGalp 99.36/5.352 70.98/5.083 77.58/4.319 67.11/4.240 ?/? ?/?
5,3,3,3,2,6,7,3,4 aLFucp 101.55/4.979 ?/3.935 ?/4.033 72.70/3.991 68.22/4.451 16.43/1.210
5,3,3,3,2,6,7,3,2 %Ac 21.98/2.210
5,3,3,3,2,6,7,3,3 %Ac 21.39/2.159
5,3,3,3,2,6,7,3 aLFucp 97.95/5.388 71.79/4.910 67.60/4.199 81.25/3.918 68.51/4.409 16.23/1.264
5,3,3,3,2,6,7 bDGlcp 103.27/4.493 74.27/3.536 83.02/3.655 69.19/3.482 77.01/3.472 61.73/3.684-3.920
5,3,3,3,2,6 aXLDmanHepp 100.36/4.893 70.96/3.973 71.99/3.818 67.16/3.859 72.49/3.607 68.27/4.221 73.07/3.856-4.057
5,3,3,3,2 aDGlcp 96.97/5.171 72.50/3.610 74.36/3.746 70.27/3.525 71.07/4.145 65.71/3.659-3.950
5,3,3,3 aDGlcp 97.93/5.462 76.60/3.678 72.05/3.850 70.28/3.541 72.45/4.068 61.02/3.819-3.906
5,3,3,6 aDGalp 99.24/5.002 69.66/3.835 70.56/3.899 70.25/4.043 ?/? ?/?
5,3,3 aDGlcp 102.56/5.197 71.35/3.663 81.42/3.941 ?/3.94 ?/4.033 65.99/3.764-4.084
5,3,4 P
5,3,7 aXLDmanHepp 100.91/4.927 ?/? ?/? ?/? ?/? ?/? ?/?
5,3 aXLDmanHepp 103.41/5.130 70.93/4.410 80.77/4.111 69.63/4.438 ?/? ?/? ?/?
5,4,0 %xXEtN 63.48/4.218 41.09/3.298
5,4 P
5 aXLDmanHepp 101.51/5.282 ?/? ?/? ?/? ?/? ?/? ?/?
aXKdo? NMR TSV error 2: unequal length of 13C and 1H datasets
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Found 1 record.
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