Cyclic beta-(1,2)-glucan synthesis in Rhizobiaceae: roles of the 319-kilodalton protein intermediate

Abstract

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...

Sequence Analysis of the Mobile Genome Island pKLC102 of Pseudomonas aeruginosa C

“…Cyclic ␤-glucan is a constitutive component of the periplasm of Agrobacterium tumefaciens, Sinorhizobium meliloti, and Brucella abortus. Knockouts of the glucan synthetase abrogate nodule formation by rhizobia (14,26), gall tumor induction by Agrobacterium (21), and intracellular multiplication of Brucella in mammalian host cells (10). The pKLC102 gene exhibits the highest homology to the A. tumefaciens and Burkholderia fungorum chvB genes.…”

Intruders below the Radar: Molecular Pathogenesis of Bartonella spp

“…Amazingly, it is apparent that the bartonellae encode the full machinery required for synthesis and secretion of cyclic ␤-(1,2)-glucans, i.e., the respective synthetase (ChvB in Agrobacterium tumefaciens [79], BH00920 in B. henselae) and the dedicated ABC exporter (ChvA in A. tumefaciens [70], BH10210 in B. henselae) (6,34). Cyclic ␤-(1,2)-glucans are circular oligosaccharides known to enforce the host interaction of both symbiotic and pathogenic Rhizobiaceae (comprehensively reviewed in reference 48).…”

Functional Mapping of Brucella abortus Cyclic β-1,2-Glucan Synthase: Identification of the Protein Domain Required for Cyclization

“…Brucella C␤G act on host cell membranes at the level of lipid rafts, controlling vacuole maturation, avoiding lysosome fusion, and allowing Brucella to reach an endoplasmic reticulum-derived vacuole permissive for bacterial replication (4). Thus, C␤G is a Brucella virulence factor required for intracellular survival; accordingly, Cgs may be a good target for developing new chemotherapy alternatives against this pathogen.Cgs catalyzes the four enzymatic activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of C␤G (1,(10)(11)(12)19). The initiation activity catalyzes the transference of a glucose residue from UDP-glucose to a not-yet-identified amino acid residue of the same protein.…”

“…Cgs catalyzes the four enzymatic activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of C␤G (1,(10)(11)(12)19). The initiation activity catalyzes the transference of a glucose residue from UDP-glucose to a not-yet-identified amino acid residue of the same protein.…”