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

Abstract

Biosynthesis of periplasmic cyclic 1,2-β-glucans inBrucella ovisstrain REO198 andB. abortusstrain S19 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 generaAgrobacteriumandRhizobium, the accumulation of the reaction products inBrucellaappeared not to be osmoticaliy regulated. Incubation of permeabilized cells with UDP-[14C]Glc led to the formation of soluble neutral cyclic 1,2-β-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 fromAgrobacterium tumefaciensA348 andRhizobium frediiUSDA191 glucoproteins known to be intermediates in the synthesis of cyclic glucans.Brucellatotal 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 inBrucellaa factor(s) that was lost or inactivated upon the preparation of membranes was required for the effective regulation between elongation and cyclization reactions.