Members of the Rhizobiaceae family synthesize cyclic -(1,2)-glucans through a mechanism which involves oligosaccharides covalently linked to a large inner membrane protein.Upon elongation to a polymer of about 15 to 25 glucose units, the oligosaccharides are cycled and thus liberated from the protein anchor. The glucose acceptor role of the inner membrane protein and the transient character of its glucosylation have been clearly demonstrated in Agrobacterium tumefaciens and Rhizobium meliloti (35), Rhizobium fredii (4, 5), Rhizobium loti (19), and all biovars of Rhizobium leguminosarum (9). After neutral cyclic -(1,2)-glucans are formed, some of them are substituted by phosphoglycerol and/or succinyl residues, probably inside the periplasmic space (3,6,13,20,21,32).The A. tumefaciens chv and R. meliloti ndv chromosomal regions code for the protein intermediates ChvB and NdvB, respectively, of approximately 319 kDa (15,33). In addition, these regions code for the ChvA/NdvA protein, which is probably involved in the transport of -(1,2)-glucans to the periplasmic space (16,23,28).It is likely that formation of cyclic -(1,2)-glucan requires at least the following three enzymatic activities: (i) one that catalyzes the transfer of the first glucose to an unknown amino acid residue in the protein intermediate, (ii) a glucosyltransferase activity responsible for chain elongation, and (iii) an activity responsible for glucan cyclization and release from the protein. Due to the fact that only cyclic glucan forms have been detected after release from the protein intermediate (34), cyclization and release reactions may proceed in the same reaction step (31,36). In this paper, we present evidence indicating that a unique protein component carries all three activities. We also suggest that this protein component is likely to be the protein intermediate. MATERIALS AND METHODSBacterial strains and culture media. A. tumefaciens and R. meliloti strains (Table 1) were grown in TY medium (0.5% tryptone and 0.3% yeast extract) and yeast extract-mannitol medium (1% mannitol, 0.1% yeast extract, 0.05% K 2 HPO 4 , 0.02% MgSO 4 and 0.02% NaCl), respectively. Bacteria were grown at 28ЊC in a rotary shaker.Inner membrane preparation. Inner membranes were purified by fractional centrifugation as previously described (24) and resuspended in 30 mM Tris-HCl buffer, pH 8.2.Native polyacrylamide gel electrophoresis. Native polyacrylamide gel electrophoresis (PAGE) was carried out in running gels of different acrylamide content (3, 5, or 7%) with an acrylamide/bisacrylamide ratio of 30:0.8, in 0.2 M Tris-HCl (pH 8.8)-0.1% Triton X-100. Gels were polymerized 20 h before electrophoresis in order to inactivate free radicals generated during polymerization. Agarose (0.7%) was added to the 3% polyacrylamide gel to improve manipulation. The stacking gels contained in all cases 3.5% acrylamide-0.1% Triton X-100-0.1 M Tris-HCl (pH 6.8) and were polymerized with 5 g of riboflavine per ml, TE-MED (N,N,NЈ,NЈ-tetramethylethylenediamine), and irradiatio...
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