IN-VIVO NUCLEAR-MAGNETIC-RESONANCE STUDY OF THE OSMOREGULATION OF PHOSPHOCHOLINE-SUBSTITUTED BETA-1,31,6 CYCLIC GLUCAN AND ITS ASSOCIATED CARBON METABOLISM IN BRADYRHIZOBIUM-JAPONICUM USDA-110

A phosphocholine-substituted beta-1,3;1,6 cyclic glucan (PCCG), an unu sual cyclic oligosaccharide, has been isolated from Bradyrhizobium jap onicum USDA 110 (D. B. Rolin, P. E. Pfeffer, S. F. Osman, B. S. Swergo ld, F. Kappler, and A. J. Benesi, Biochim, Biophys. Acta 1116:215-225, 1992). Data presented here suggest that PCCG synthesis is dependent o n the carbon metabolism and that osmotic regulation of its biosynthesi s parallels regulation of membrane-derived oligosaccharide biosynthesi s observed in Escherichia cell (E. P. Kennedy, M. K Rumley, H. Schulma n, and L. M. G. van Golde, J. Biol. Chem. 251:4208-4213, 1976) and Agr obacterium tumefaciens (G. A. Cangelosi, G. Martinetti, and E. W. Nest er, J. Bacteriol. 172:2172-2174, 1990). Growth of B. japonicum USDA 11 0 cells in the reference medium at relatively low osmotic pressures (L O) (65 mosmol/kg of H2O) caused a large accumulation of PCCG and unsub stituted beta-1,3;1,6 cyclic glucans (CG). Sucrose and polyethylene gl ycol, nonionic osmotica, reduce all growth rates and inhibit almost co mpletely the production of PCCG at high osmotic pressures (HO) above 6 50 and 400 mosmol/kg of H2O), respectively. We used in vivo C-13 nucle ar magnetic resonance spectroscopy to identify the active osmolytes im plicated in the osmoregulation process. The level of alpha,alpha-treha lose in B. japonicum cells grown in autoclaved or filter-sterilized so lutions remained constant in HO (0.3 M sucrose or 250 g of polyethylen e glycol 6000 per liter) medium. Significant amounts of glycogen and e xtracellular polysaccharides were produced only when glucose was prese nt in the autoclaved HO 0.3 M sucrose media. The results of hypo- and hyperosmotic shocking of B. japonicum USDA 110 cells were monitored by using in vivo P-31 and C-13 nuclear magnetic resonance spectroscopy. The first observed osmoregulatory response of glycogen-containing cell s undergoing hypoosmotic shock was release of P-i into the medium. Wit hin 7 h, reabsorption of P-i was complete and production of PCCG was i nitiated. After 12 h, the PCCG content had increased by a factor of 7. Following the same treatment, cells containing little or no glycogen released trehalose and failed to produce PCCG. Thus the production of PCCG/CG in response to hypoosmotic shocking of stationary-phase cells was found to be directly linked to the interconversion of stored glyco gen. Hyperosmotic shocking of LO-grown stationary-phase cells with suc rose had no effect on the content of previously synthesized CG/PCCG. T he PCCG/CG content and its osmotically induced biosynthesis are discus sed in terms of carbon metabolism and a possible role in hypoosmotic a daptation in B. japonicum USDA 110.