Three Enzymatic Steps Required for the Galactosamine Incorporation into Core Lipopolysaccharide

Abstract

The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (␣HexN-(1,4)-␣GalA) attached by an ␣1,3 linkage to L-glycero-D-manno-heptopyranose II (L-glycero-␣-D-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is D-glucosamine, whereas in other P. mirabilis strains, it corresponds to D-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of D-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of D-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase.

Phage Resistance in Multidrug-Resistant Klebsiella pneumoniae ST258 Evolves via Diverse Mutations That Culminate in Impaired Adsorption

“…The waa gene cluster (homologous to rfa in E. coli) encompasses genes that synthesize and modify the hexose region of the lipopolysaccharide (LPS) core in stepwise fashion (26,27). WaaC is involved in the addition of HepI to KdoI of the inner core and WaaF in the addition of HepII to HepI, and WaaQ appears to be involved in the addition of HepIII to the outer core (28,29). All three are presumed heptosyltransferases, and null mutations in cognate gene rfaC or rfaF yield deep, rough mutants in E. coli and S. Typhimurium (27,30).…”

“…The gene wabH, an rfaG homologue, constitutes part of the same locus. WabH catalyzes the transfer of GlcNAc from UDP-GlcNAc to the outer core, and wabH mutants also produce truncated LPS (29,31).…”

Genomic and Proteomic Studies on Plesiomonas shigelloides Lipopolysaccharide Core Biosynthesis

“…An alignment between these two glycosyltransferases (WaaO, Q8KMW9; WaaI, Q9ZIT4) revealed that they share 51 and 80% amino acid residue identity and similarity, respectively. These levels of identity/similarity are similar to the ones shared by the P. mirabilis N-acetylglucosaminyl (WabH R110 ) and N-acetylgalactosaminyl (WabP 50/57 ) transferases (42). P. shigelloides WapE is a UDP-galactose transferase ␤-(1¡4) to L-HepI instead of the UDP-glucose transferase ␤-(1¡4) to LHepI characteristic of the Klebsiella-Serratia-Proteus group.…”

“…We previously showed that the incorporation of GlcN in the LPS core requires two distinct enzymatic steps due to the lack of UDP-GlcN in prokaryotes (39,42). First, one enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer-core LPS, and then a second enzyme catalyzes the deacetylation of the core LPS containing the GlcNAc residue.…”

Functional Identification of Proteus mirabilis eptC Gene Encoding a Core Lipopolysaccharide Phosphoethanolamine Transferase

“…Some P. mirabilis core-OS structures contain unusual residues such as quinovosamine, an open-chain form of N -acetyl-galatosamine, or amide linked amino acids. Recently, most of the genes involved in the biosynthesis of the sugar backbone of the core LPS from several P. mirabilis strains have been identified and characterized [ 10 , 11 ]. Little is known about the role of the non-sugar charged residues or groups in the core OS.…”