Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B‐band lipopolysaccharide gene cluster

Abstract

Pseudomonas aeruginosa co-expresses A-band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B-band LPS, a heteropolymer with a repeating unit of 2-5 sugars which is the serotype-specific antigen. The gene clusters for A- and B-band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B-band O-antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz (rol), wzy (rfc), and wbpA-wbpN. A further 3 ORFs not thought to be involved with LPS synthesis were identified (hisH, hisF, and uvrB). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O-antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup-specific, knockout mutations confirmed it is necessary for O5 O-antigen biosynthesis. A novel insertion sequences, IS 1209, is present at the junction between the serogroup-specific and non-specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O-antigen biosynthesis.

Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens

“…An IS element (ISAS1) is also responsible for mutation to O-antigen deficiency in some isolates of Aeromonas salmonicida (8). An IS element has also been found within the rfb gene cluster of Pseudomonas aeruginosa O5, although it does not inactivate O-antigen expression (7). Pairs of insertion sequences, on the other hand, may lead In several members of the Enterobacteriaceae, such as S. enterica serovar Typhimurium (4), E. coli O111 (4), and Shigella flexneri 2a (31), which all produce heteropolysaccharide O antigens, the gene order in the his-gnd region of the chromosome is conserved: his-wzz-ugd-gnd-rfb.…”

Role of glycan synthesis in colonization of the mammalian gut by the bacterial symbiont Bacteroides fragilis

“…UngD1 and UngD2 each contain the Ser-Met-Lys catalytic triad, the GxxGxxG nucleotide co-factor binding motif, and the conserved glutamate residue characteristic of this branch of the short-chain dehydrogenase/reductase family of enzymes (12). To determine if ungD1 and ungD2 encode UDP-GlcNAc 4,6 dehydratase activity, we assessed the ability of each to complement a wbpM mutant strain of P. aeruginosa PAO1 (13). Each was able to restore expression of serotype O5 LPS to P. aeruginosa wbpM::Gm (Fig.…”

Biochemical Characterization of WbpA, a UDP-N-acetyl-d-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1

“…We previously proposed that biosynthesis of the mannosaminuronic acid-derived residues of P. aeruginosa would follow a similar pathway to UDP-D-ManNAcA biosynthesis based on ϳ50% amino acid similarity between WecB and WbpI, and between WecC and WbpA (9,15). The current investigation of the activity of WbpA was prompted by the inability of wbpI and wbpA to complement wecB and wecC knockout mutants (15).…”

“…These three sugar residues are postulated to be synthesized from the common precursor UDP-N-acetyl-D-glucosamine (UDP-D-GlcNAc) (9), and the initial steps in the biosynthesis of the fucosamine moiety have previously been described (10,11). N-Acetyl-D-mannosaminuronic acid residues are present in both the enterobacterial common antigen (ECA) of Escherichia coli and the polysaccharide capsule of Staphylococcus aureus, and the biosynthesis of the nucleotide-activated precursor to these molecules, UDP-N-acetyl-D-mannosaminuronic acid (UDP-D-ManNAcA), is synthesized from UDP-DGlcNAc via a two-step pathway.…”