Synthesis of glycerophosphorylated cyclic (1,2)- -glucans in Rhizobium meliloti strain 1021 after osmotic shock

Abstract

The transfer of phosphoglycerol moieties from phosphatidylglycerol to the cyclic (1,2)-/?-glucans in growing cultures of Rhizobium meliloti strain 1021 was investigated using pulse-chase experiments with [3H]glycerol and/or [14C]glucose. No transfer occurred when cells were grown and pulse-chased in a medium containing 04 M NaCl. However, radiolabelled glycerophosphorylated cyclic (1,2)-/?-glucans could be detected within 30 min after transfer of these cultures to a low-osmolarity medium. Conversely, when low-osmolarity cultures were shifted to a high-osmolarity medium containing 04 M NaCl or 0 8 M sucrose, the transfer of phosphoglycerol substituents to the cyclic (1,2)-/3-glucans was inhibited. Further experiments revealed that the transfer of phosphoglycerol substituents to the cyclic (1,2)-/3-glucans occurs within the periplasmic compartment.

A novel cyclic beta-1,2-glucan mutant of Rhizobium meliloti

“…Because mutant S9 is unable to transfer phosphoglycerol substituents to its cyclic ␤-1,2-glucans, studies with this mutant should provide important insight concerning the succinyl transferase reaction. For example, we recently have shown that the transfer of phosphoglycerol substituents to the cyclic ␤-1,2-glucan backbone occurs in the periplasm (5). These experiments involved pulse-labeling cultures with [ 14 C]glucose coupled with the selective release of periplasmic cellular constituents by polymyxin B treatment (5).…”

“…We have previously shown that the transfer of phosphoglycerol substituents to the cyclic ␤-1,2-glucans may be measured in vivo after pulse labeling R. meliloti cultures with [ 3 H]glycerol (5,27). Using this approach, we were unable to detect the transfer of [ 3 H]phosphoglycerol substituents to the cyclic ␤-1,2-glucans of mutant S9 (e.g., the rate was less than 4% of that measured within R. meliloti 1021 cultures) (data not shown).…”

“…The dimensions of the column were 1 by 17 cm. Fractions (2 ml) were collected, and aliquots were assayed for total carbohydrate by the phenol-sulfuric acid method (3). N, neutral cyclic ␤-1,2-glucans; I to VI, anionic cyclic ␤-1,2-glucan subfractions.…”

“…The two triplets between 2.4 and 2.8 ppm correspond to the methylene protons of the succinic acid substituents (3). The assignment of the other major resonances has been described previously (3).…”

“…In Agrobacterium and Rhizobium species, these molecules contain glucose residues linked solely by ␤-1,2-glycosidic bonds, and the predominant ring size distribution is between 17 and 25 glucose residues (4). Additionally, these glucans may become highly modified with anionic substituents such as phosphoglycerol and/or succinyl moieties (1,3,27,30). For example, the anionic cyclic ␤-1,2-glucans of Rhizobium meliloti carry, on average, a total net negative charge of approximately 2.5, and some cyclic ␤-1,2-glucan subfractions synthesized by this bacterium contain as many as six anionic substituents (6).…”