The Incorporation of Glucosamine into Enterobacterial Core Lipopolysaccharide

Abstract

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide ␣GlcN-(1,4)-␣GalA attached by an ␣1,3 linkage to L-glycero-D-manno-heptopyranose II (LD-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of D-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide ␣GlcN-(1,4)-␣GalA attached by an ␣1,3 linkage to LD-HeppII.

Three Enzymatic Steps Required for the Galactosamine Incorporation into Core Lipopolysaccharide

“…Compositional analysis of this fraction by GC-MS of acetylated methyl glycosides revealed the presence of Kdo, L-glycero-D-manno-heptopyranose, GalA, Glc, and GalN. Comparative MALDI-TOF analysis of this core OS fraction with that of strain 52145⌬waaL wabH (15) revealed major signals at 1488.19 and 1470.27 m/z, corresponding to a Kdo-Hep3-HexHexA2-HexN and its anhydro form. This is in agreement with a core extending up to the second outer OS residue (GalN) (Fig.…”

“…The LPS from K. pneumoniae 52145⌬wabN is devoid of the last two outer core residues and O-PS and presents GlcNAc instead of GlcN (15). Introduction of pGEM-T-wabN R110 or pGEM-T-wabN 50/57 restores wild type 52145 LPS migration and O-PS production (Fig.…”

“…11). This common fragment is also found in the core LPS of Klebsiella pneumoniae and Serratia marcescens (12)(13)(14), and we have shown that two enzymes are required for the incorporation of GlcN into the core LPS of K. pneumoniae: the WabH enzyme catalyzing the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS and WabN catalyzing the deacetylation of the incorporated GlcNAc (15). The rest of the Proteus core OS is quite variable, with up to 37 different structures recognized in the genus Proteus and 11 in P. mirabilis (11).…”

“…A, the common part of core OS shared by all of the studied strains of P. mirabilis (11), K. pneumoniae, and S. marcescens (11,13,14). Broken lines denote the truncation level for the different core biosynthetic gene mutations (13,14,15,16,35). B, the core OS structures of P. mirabilis strains 50/57 and TG83 (11) wabP 50/57 ).…”

“…WabN Deacetylates Both GlcNAc and GalNAc LPS in VitroAn in vitro enzymatic assay measuring the amount of radiolabeled GlcNAc or GlcN incorporated into acceptor LPS from UDP-[ 14 C]GlcNAc after acid hydrolysis and TLC separation of the radiolabeled residues was performed (15). LPS from mutant 52145⌬wabH was used as an acceptor with combinations of histidine-tagged enzymes WabH, WabP, and WabN.…”