Abstract
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.
Highlights
The network of E. coli surface polysaccharides can be further subdivided into those that are tightly associated or covalently linked to the outer membrane (OM) (O/K-antigens, ECA) and those that are loosely associated, called exo- or slime polysaccharides
The genetic determinant for Colanic acid (CA) biosynthesis resides on the 19 gene wca cluster [12, 13] and is tightly regulated by a complex signal transduction cascade governed by the rcs phosphorelay system [14, 15]
A unifying theme among CA-inducing stresses is that they all disturb the integrity of the cell envelope, leading to the suggestion that CA synthesis is a response to sensed OM instability originating from a damaged cell envelope [16]
Summary
BW30270 F11119-41 TCM31 TCM33 TCM40 KPM22 KPM25 KPM72 KPM73 KPM77 pT7waaL pT7wbbL. E. coli K-12 MG1655; rphϩ fnrϩ wbbL Wild-type E. coli isolate with O:16 antigen; K1:HϪ. BW30270 ⌬waaL TCM31(pT7waaL) BW30270(pT7wbbL); O:16ϩ TCM15 derivative; LPSϪ KPM22 with pT7kdsD. KPM22 ⌬waaL KPM72(pT7waaL) KPM22(pT7wbbL); O:16ϩ pT7-7 with E. coli K-12 waaL; AmpR pT7-7 with E. coli F11119-41 wbbL; AmpR. A mutant strain of E. coli K-12 that produces copious quantities of CA when cultured in hypotonic growth medium has been used to isolate a novel LPS glycoform containing CA repeats that we call MLPS (for M-antigen). It is shown that MLPS has a structure differing from the reported structures of extracellular CA and that MLPS, not LPS O-antigen, is the dominant smooth LPS glycoform synthesized when CA is highly induced
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