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

Abstract

Biosynthesis of periplasmic cyclic 1,2-beta-glucans in Brucella ovis strain REO198 and B. abortus strain 519 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]Glc led to the formation of soluble neutral cyclic 1,2-beta-glucans and [14C]glucose-labelled 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.