Eleven of the 20 International Antigenic Typing Plan (IATS) serotypes produce LPSs that lack the terminal d-glucose residue (GlcIV). of a terminal d-glucose in these core OS structures. Our results strongly suggested that encodes a 1,2-glucosyltransferase. INTRODUCTION is usually ubiquitous in the environment and generally regarded as a saprophyte, but it is also an important opportunistic human and animal pathogen (34). This bacterium can cause a variety of infections including some unusual ones, such as green nail syndrome associated with the use of community pools (17) and keratitis associated with the use of contact lenses (37), but mainly it infects compromised individuals, such as AIDS patients and those with burn wounds and cystic fibrosis (CF). For CF patients, is the major cause of morbidity and mortality (11, 32). is usually a Gram-negative bacterium and possesses lipopolysaccharide (LPS) as a major constituent of the outer leaflet of the outer membrane. LPS also serves as one of its major virulence factors (7, 30). Due to its proximity in the bacterial outer envelope, LPS plays crucial functions in maintaining structural integrity and interacting with the environment. LPS is composed of three distinct regions: (i) lipid A, the endotoxic moiety that anchors the LPS molecule in the outer membrane; (ii) the core oligosaccharide (OS); and (iii) the long-chain O polysaccharides (or O antigen) that consist of different repeated sugar models. These features segregate strains into 20 International Antigenic Typing Plan (IATS) serotypes. The core oligosaccharide (OS) can be divided into inner and outer regions. The inner core is usually conserved among Gram-negative bacteria and is composed of two 3-deoxy-d-cell simultaneously produces two unique core OS glycoforms. The first glycoform is usually capped, meaning that it is covalently attached to O antigen, while the second, uncapped core is usually devoid of O antigen. Besides the presence or absence of an O antigen, the two core glycoforms differ in the outer core region, particularly in the position and linkage of L-Rha, and in the presence/absence of a terminal Glc residue (GlcIV) (Fig. 1). The basic core OS structure is usually conserved among different strains; however, variations can be observed in the presence of GlcIV in uncapped outer core and noncarbohydrate substituents (such as phosphorylation of Hep residues or acetylation on certain sugar residues of the core). Immunochemical data produced by our group (9) and the structural elucidation of core OS reported by Bystrova et al. (2) revealed that only 9 out of the 20 IATS serotypes (O2, O5, O7, O8, O10, O16, O18, O19, and O20) reacted with outer core-specific monoclonal antibody (MAb) 5c-101, and the elucidated core OS structures of these serotypes possess terminal GlcIV. Even though chemical structures of the core OS of the wild-type PAO1 strain and the IATS serotypes (2) have been elucidated, knowledge of how outer core residues are transferred to synthesize the core is usually lacking. We reported earlier that MigA and WapR are two putative rhamnosyltransferases associated with outer core OS biosynthesis, and these two enzymes share 35% identity. Analysis of LPS from mutant has not been decided, and a mutant could not be constructed even though various strategies were used (23). Apparently, is an essential gene, and mutation in this gene is usually lethal. Based on homology to MigA and WapR, we hypothesized that the product of the uncharacterized gene is also involved in outer core OS biosynthesis. To conform to the widely accepted LPS gene nomenclature and be consistent with other genes associated with core OS biosynthesis as as serotypes. Open in a separate windows Fig. 1. Structures of the two unique outer core OSs that are simultaneously produced by a single PAO1 cell. (A) Uncapped core OS is usually devoid of O antigen and contains an -1,6-linked l-Rha and 1, TGR-1202 2-linked TGR-1202 d-GlcIV. (B) Capped core OS is usually has a substitution of O polysaccharide through an -1,3-linked l-Rha. GalN, galactosamine; Ala, alanine; Rha, rhamnose; Glc, glucose. Putative glycosyltransferases MigA, WapR, and WapB required for transfer of RhaA, RhaB, and GlcIV, respectively, are depicted by arrowheads (adapted from recommendations 18 and 26). MATERIALS AND METHODS Bacterial strains and culture conditions. The bacterial strains and plasmids used in this study are outlined in Table 1. Bacterial strains were produced in lysogeny broth (LB; also commonly known TGR-1202 as Luria-Bertani medium) (Invitrogen) LEPR at 37C. Antibiotics were used at the following concentrations: for strains, 100 g/ml ampicillin, 15 g/ml gentamicin, and 15 g/ml tetracycline; and for strains, 300 g/ml carbenicillin, 150 g/ml.
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