Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta-or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo 2 Hex 6 GalA 3 Fuc3NAcRha 4 and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.

Cyclic ␤-1,2-glucans (C␤G) are osmolyte homopolysaccharides with a cyclic ␤-1,2-backbone of 17-25 glucose residues present in the periplasmic space of several bacteria. Initiation, elongation, and cyclization, the three distinctive reactions required for building the cyclic structure, are catalyzed by the same protein, the C␤G synthase. The initiation activity catalyzes the transference of the first glucose from UDP-glucose to a yet-unidentified amino acid residue in the same protein. Elongation proceeds by the successive addition of glucose residues from UDP-glucose to the nonreducing end of the protein-linked ␤-1,2-oligosaccharide intermediate. Finally, the protein-linked intermediate is cyclized, and the cyclic glucan is released from the protein. These reactions do not explain, however, the mechanism by which the number of glucose residues in the cyclic structure is controlled. We now report that control of the degree of polymerization (DP) is carried out by a ␤-1,2-glucan phosphorylase present at the C␤G synthase C-terminal domain. This last activity catalyzes the phosphorolysis of the ␤-1,2-glucosidic bond at the nonreducing end of the linear protein-linked intermediate, releasing glucose 1-phosphate. The DP is thus regulated by this ''lengthcontrolling'' phosphorylase activity. To our knowledge, this is the first description of a control of the DP of homopolysaccharides.cyclic ␤-1,2-glucan ͉ phosphorylase ͉ size control O smoregulated periplasmic glucans are oligosaccharides present in the periplasm of certain Gram-negative bacteria. Common features of these oligosaccharides are the presence of glucose as the sole sugar constituent and the regulation of their synthesis by the osmolarity of the growth media. Osmoregulated periplasmic glucans may be cyclic, branched cyclic, or branched linear and, depending on the species, may be substituted with a variety of nonglycosidic residues (1, 2). Agrobacterium, Rhizobium, Sinorhizobium, and Brucella species synthesize osmoregulated periplasmic glucans of family II. Glucans of this family have 17-25 glucose residue cyclic ␤-1,2-backbones substituted with sn-1-phosphoglycerol, succinic acid, methylmalonic acid, or a combination of them (1-3).Cyclic ␤-1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of cyclic ␤-1,2-glucans (C␤G), is present in a restricted number of symbiotic or pathogenic bacteria, most of them belonging to the ␣-proteobacteria group, in which C␤G are a symbiotic or virulence factor required for successful host interaction (4-9). Brucella abortus Cgs is a 320-kDa (2,867 amino acid residues) polytopic integral inner membrane protein with six transmembrane-spanning segments (TMSs) and with the N and C termini located on the cytoplasmic side of the membrane (10). Cgs, an enzyme using UDP-glucose as sugar donor and Mg 2ϩ as cofactor, functions as an inverting processive ␤-1,2-glucosyltransferase that catalyzes the three enzymatic activities (initiation, elongation, and cyclization) required for synthesis of C␤G. Synthesis is initiated...