Molecular cloning and characterization of a locus responsible for O acetylation of the O polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide

Abstract

Complementation experiments, Tn5 mutagenesis, and DNA sequencing were used to identify a locus (lag-1) that participates in acetylation of Legionella pneumophila serogroup 1 lipopolysaccharide. Nuclear magnetic resonance analyses of lipopolysaccharides from mutant and complemented strains suggest that lag-1 is responsible for O acetylation of serogroup 1 O polysaccharide.

FIG. 1

Structure of L. pneumophila serogroup 1 LPS adopted from Knirel et al. (–11), Zähringer et al. (24), and Moll et al. (18). (A) Schematic representation of the whole LPS structure. (B) Structure of OPS. Sugar abbreviations: GlcN3N, 2,3-diamino-2,3-dideoxy-d-glucose; Kdo, 3-deoxy-d-manno-octulosonic acid; Leg and iso-Leg, derivatives of legionaminic acid and its C4 epimer, respectively; Rha, l-rhamnose; QuiNAc, 2-acetamido-2,6-dideoxy-d-glucose (N-acetylquinovosamine); R = Ac in strains Philadelphia 1 and CS338 or R = H in strain CS332.

FIG. 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (A) and Western blot (B) analyses of LPSs from Philadelphia 1 (wild-type) and lag-1 mutant strains. (A) LPS was isolated as described previously (19), resolved on a sodium dodecyl sulfate–14% polyacrylamide gel, and visualized by silver staining. Equal amounts of LPS (∼1.0 μg) were added to each lane. Lanes: 1, S. enterica serovar Typhimurium; 2, Philadelphia 1; 3, CS332 (lag-1 negative); 4, CS334 (CS332/pLPS16 [lag-1 positive]). (B) Equal amounts of LPS from strains Philadelphia 1, CS332, and CS338 (CS332/pLPS17 [lag-1 positive]) were resolved on sodium dodecyl sulfate–14% polyacrylamide gels, transferred to nitrocellulose paper, and probed with MAB2 as described by Mintz and Zou (15). Lanes: 1, Philadelphia 1; 2, CS332; 3, CS338.

FIG. 3

Localization of lag-1 by Tn5 mutagenesis. Tn5 insertions were introduced into pLPS16.1 and mapped according to the methods of de Bruijn and Lupinski (2). Each triangle represents the position of a Tn5 insertion in pLPS16.1. Plasmids containing each of the Tn5 insertions were electroporated into strain CS332, and transformants were tested for the ability to bind MAB2, 33G2, and 144C2. (−), insertions eliminating binding of MAB2 and 33G2; (+), insertions having no effect on the binding of MAB2 and 33G2. Wild-type LPS has the following MAb binding pattern: MAB2 (+), 33G2 (+), 144C2 (−). Restriction sites: C, ClaI; E, EcoRI; H, HindIII; S, SphI; X, XhoI. The black box defines the physical location of the lag-1 locus as determined by DNA sequencing experiments.

FIG. 4

¹³C-NMR spectra and corresponding structures of OPSs from LPSs produced by wild-type, mutant, and complemented strains. (A) CS338 (CS332/pLPS17). (B) CS339 (CS332/pLPS20). (C) CS333 (AM511/pLAW300). Signals for the 8-O-acetyl group (Me at δ 22.0 and CO at δ 174.4) are marked by stars. Numbers refer to carbons C1 to C9 of legionaminic acid.