Periplasmic cyclic 1,2-β-glucan in Brucella spp. is not osmoregulated

Abstract

Biosynthesis of periplasmic cyclic 1,2-p-glucans in Brucella ovis strain RE01 98 and B. abortus strain Sl9 was found to be carried out by membrane-bound enzymes that use UDP-glucose (UDP-Glc) as donor substrate. Contrary to what happens in species of the genera Agrobacterium and Rhizobium, the accumulation of the reaction products in Brucella appeared not to be osmotically regulated. Incubation of permeabilized cells with UDP-[14C]GI~ led to the formation of soluble neutral cyclic 1,2-p-glucans and [14C]glucoselabelled glucoproteins. PAGE of pulse-chase experiments carried out with permeabilized cells showed that the molecular mass of the labelled protein was indistinguishable from Agrobacterium tumefaciens A348 and Rhizobium fredii USDA191 glucoproteins known to be intermediates in the synthesis of cyclic glucans. Brucella total membrane preparations were less efficient than permeabilized cells in the formation of cyclic glucan; this was attributed to defective cyclization. Accumulation of protein intermediates having oligosaccharides of high molecular mass that were not released from the protein was observed after chase with 2 mM UDP-Glc. This defect was not observed when permeabilized cells were used as enzyme preparation, thus suggesting that in Brucella a factor(s) that was lost or inactivated upon the preparation of membranes was required for the effective regulation between elongation and cyclization reactions.

The Brucella abortus Cyclic β-1,2-Glucan Virulence Factor Is Substituted with O-Ester-Linked Succinyl Residues

“…1A). The elution volume of this material corresponds to the previously characterized cyclic ␤-1,2-glucans of B. abortus (10). Cellular ␤-1,2-glucans recovered from the Bio-Gel P4 column (fractions 13 to 22) were subjected to DEAE-Sephadex chromatography (Fig.…”

“…The ethanolic extracts were centrifuged in an Eppendorf centrifuge, and the supernatants were dried in a speed-vac centrifuge. The extracted glucans were dissolved in 70% ethanol and subjected to thin-layer chromatography (TLC) as described previously (10). The TLC plates were developed by spraying them with 5% sulfuric acid in ethanol and heating them for 10 min at 120°C.…”

“…Virulence was determined by quantitating the survival of the strains in mouse spleens at different times postinfection, as previously described (16). Groups of 9-week-old female BALB/c mice were injected intraperitoneally with 10 4 CFU of B. abortus 2308, B. abortus S19, and their isogenic mutants B. abortus Cgm08 and B. abortus Cgm19 in 0.2 ml of sterile PBS. At 2, 4, and 12 weeks postinfection, the animals were sacrificed, and the spleens were removed, weighed, homogenized in sterile 150 mM NaCl solution, serially diluted, and plated onto BB agar with the appropriate antibiotics to determine the number of CFU per spleen.…”

A glycosyltransferase with a length-controlling activity as a mechanism to regulate the size of polysaccharides

“…Preparation of total membranes of A. tumefaciens strains was carried out as described previously (11). Protein concentration was determined by the method of Lowry et al (20).…”

“…Elongation is catalyzed by a UDP-Glc:␤-1,2-oligosaccharide glucosyltransferase activity responsible for the addition of glucose residues to the nonreducing end of the linear ␤-1,2-oligosaccharide linked to the protein. Finally, cyclization, which is coupled to the release of the C␤G from the protein, is probably a transglucosylation reaction during which the nonreducing end of the protein-linked oligosaccharide forms a ␤-1,2 linkage with the protein-linked reducing end of the polyglucose chain (6,11).…”