Arch Microbiol (1995) 163 : 454-462 9 Springer-Verlag 1995 Olga A. Castro 9 Angeles Zorreguieta Carlos Semino - Luis Ielpi Biosynthesis of cyclic -(1,2)-glucans in Rhizobium leguminosarum biovars viciae, phaseoli and trifolii Received: 9 February 1995 / Accepted: 1 March 1995 Abstract Inner membranes of Rhizobium leguminosarum biovars viciae, phaseoli, and trifolii synthesized a bet- erogenous family of neutral cyclic [3-(1,2)-glucans in a re- action system that used oligosaccharide intermediates co- valently bound to a large protein. This glucoprotein showed a slightly lower mobility on SDS-polyacrylamide gels (apparent mol. mass of 320 kDa) than the [3-(1,2)-glu- can intermediate protein of Rhizobium meliloti. In vivo pulse-label experiments with growing cells of R. legumi- nosarum biovar trifolii RS800 using radioactive glucose showed that few species of cyclic [3-(1,2)-glucans were synthesized and up to 30% were substituted with charged non-glycosidic residues, probably sn-l-phosphoglycerol. Key words Rhizobium leguminosarum biovars 9 Biovar trifolii. Biovar phaseoli . Biovar viciae 9 Cyclic [3-(1,2)- glucan - Glucoprotein intermediate 9 Polysaccharide synthesis Introduction Members of the Rhizobiaceae family produce cyclic [3- (1,2)-glucans that are proposed to play a role in plant in- fection (Puvanesarajah et al. 1985; Geremfa et al. 1987; St anfield et al. 1988). In Agrobacterium tumefaciens and Rhizobium meliloti, the biosynthesis of [3-(1,2)-glucans has been extensively studied (Amemura 1984; Zorregui- eta et al. 1985a, b; Zorreguieta and Ugalde 1986). [3-(1,2)- Glucooligosaccharide-protein intermediates are formed O.A. Castro 9 A. Zorreguieta ([g~) 9 C. Semino I 9 L. Ielpi Instituto de Investigaciones Bioqufmicas "Fundaci6n Campomar", Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires and CONICET, Avenida Patricias Argentinas 435, 1405 Buenos Aires, Argentina Fax + 54-1-865-2246; e-mail angelesz@iibfc.uba.ar Present address: 1Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, USA (Zorreguieta et al. 1985b; Zorreguieta and Ugalde 1986). The glucose acceptor is a large inner membrane protein, with an apparent molecular mass of 235kDa (Zorreguieta and Ugalde 1986) encoded by the chvB and the ndvB genes in A. tumefaciens and R. meliloti, respectively (Dy- lan et al. 1986; Zorreguieta et al. 1988; Ielpi et al. 1990). Ring size and relative amount of each glucan depend on the species (Amemura 1985; Zevenhuizen et al. 1990). The in vivo synthesis of glucans in R. leguminosarum biovars is different than that of R. meliloti and A. tumefa- ciens. It has been proposed that periplasmic [3-(1,2)-glu- can protects cells of R. meliloti and A. tumefaciens against hypoosmotic stress. Furthermore, synthesis of periplasmic cyclic ~-(1,2)-glucans is strongly repressed in A. tumefa- ciens (Miller et al. 1986; Zorreguieta et al. 1990) and R. meliloti (Dylan et al. 1990) in medium with high osmotic strength. In contrast, large quantities of glucans are ex- creted from cells of R. leguminosarum biovar trifolii TA- 1, while the cellular glucan content does not change at high NaC1 concentration (Breedveld et al. 1991, 1992). In this paper, we examined the in vitro [3-(1,2)-glucans synthesis in R. leguminosarum biovars viciae, phaseoli, and trifolii. We analyzed the reaction intermediates and compared them to those of R. meliloti and A. tumefaciens. In addition, the glucans synthesized in vivo by R. legumi- nosarum biovar trifolii were analyzed. Materials and methods Organisms and culture media The following strains were used: A. tumefaciens A 348 (Douglas et al. 1985), A. tumefaciens ME 3 (Douglas et al. 1985), R. meliloti 102F34 (Ditta et al. 1980), and R. Ieguminosarum biovars trifoli: Rs800 (Megias et al. 1982), phaseoli 8002 (Lamb et al. 1982), and viciae 128C53 (Brewin et al. 1980). Strains were grown in either yeast extract-mannitol medium (1% mannitol, 0.1% yeast extract, 0.05% K2HPO 4, 0.02% Mg(SO4)2 and 0.02% NaC1) or in TY medium (0.5% tryptone and 0.3% yeast extract). Cultures were grown at 28~ on a rotary shaker.