Functional genomics of Helicobacter pylori: identification of a β‐1,4 galactosyltransferase and generation of mutants with altered lipopolysaccharide

Abstract

A previously annotated open reading frame (ORF) (HP0826) from Helicobacter pylori was cloned and expressed in Escherichia coli cells and determined to be a β‐1,4‐galactosyltransferase that used GlcNAc as an acceptor. Mutational analysis in H. pylori strains demonstrated that this enzyme plays a key role in the biosynthesis of the type 2 N‐acetyl‐lactosamine (LacNAc) polysaccharide O‐chain backbone, by catalysing the addition of Gal to GlcNAc. To examine the potential role of this O‐chain structure in bacterial colonization of the host stomach, the mutation was introduced into H. pylori strain SS1 which is known to be capable of colonizing the gastric mucosa of mice. Compared with the parental strain, mutated SS1 was less efficient at colonizing the murine stomach.

Microwave-assisted sample preparation for rapid and sensitive analysis of H. pylori lipid A applicable to a single colony

“…We have previously confi rmed that H. pylori 26695/hp0479::kan expressing a truncated LPS was defi cient in its ability to successfully colonize the murine stomach ( 23 ). We have also constructed H. pylori 26695/hp0826::kan mutant and demonstrated that this mutation resulted in formation of truncated LPS lacking the O-chain polysaccharide but maintaining the outer core region ( 26 ). Finally, we have previously described 26695/hp1191::kan mutant expressing a deep-rough inner core LPS structure lacking both the O-chain and the outer core region and containing a single Kdo residue attached to LD-heptose residue substituted by a P Etn group.…”

Identification of a d - glycero - d - manno -Heptosyltransferase Gene from Helicobacter pylori

“…Mutagenesis of the cloned HP0479 gene of pHP0479G.1 was carried out by the insertion of a kanamycin resistance marker from C. coli (24) as previously described (29), generating plasmid pHP0479G.1kan.…”

“…Membrane fraction analysis. Membrane fractions were prepared from overnight (18-h) liquid cultures of HP0479 mutant strains and parental strains by using the protocols described by Logan and coworkers (29). The fractions were analyzed by one-dimensional SDS-PAGE and stained using Coomassie blue.…”

Enzymatic synthesis of vancomycin derivatives using galactosyltransferase and sialyltransferase

“…It was cloned nearly a decade ago and demonstrated that HP0826 has a highly efficient activity for the synthesis of trisaccharides (Gala1, 4Galb1, 4GlcNAc) with recombinant bacteria. [9][10][11] Another glycosyltransferase gene, Pm0188, which encodes multifunctional sialyl transferases (SiaTs), was cloned from Pasteurella multocida genomic strain Pm70 and four functions, such as a2,3-SiaT, a2,6-SiaT, sialidase and trans-sialidase, have been identified. 12 Especially, a2,3-SiaT can transfer a sialic acid (Sia) residue from CMP-Sia to galactosides to form a2,3 sialyl linkages efficiently at a wide range of pH values (pH 7.5-9.0).…”