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...

No abstract

Calcium-activated anion secretion is expected to ameliorate cystic fibrosis, a genetic disease that carries an anion secretory defect in exocrine tissues. Human patients and animal models of the disease that present a mild intestinal phenotype have been postulated to bear a compensatory calcium-activated anion secretion in the intestine. TMEM16A is calcium-activated anion channel whose presence in the intestinal epithelium is contradictory. We aim to test the functional expression of TMEM16A using animal models with Cftr and/or Tmem16a intestinal silencing. Expression of TMEM16A was studied in a wild type and intestinal Tmem16a knockout mice by mRNA-seq, mass-spectrometry, q-PCR, Western blotting and immunolocalization. Calcium-activated anion secretion was recorded in the ileum and proximal colon of these animals including intestinal Cftr knockout and double mutants with dual Tmem16a and Cftr intestinal ablation. Mucus homeostasis was studied by immune-analysis of Mucin-2 (Muc2) and survival curves were recorded. Tmem16a transcript was found in intestine. Nevertheless, protein was barely detected in colon samples. Electrophysiological measurements demonstrated that the intestinal deletion of Tmem16a did not change calcium-activated anion secretion induced by carbachol or ATP in ileum and proximal colon. Muc2 architecture was not altered by Tmem16a silencing as was observed when Cftr was deleted from mouse intestine. Tmem16a silencing neither affected animal survival nor modified the lethality observed in the intestinal Cftr -null mouse. Our results demonstrate that TMEM16A function in the murine intestine is not related to electrogenic calcium-activated anion transport and does not affect mucus homeostasis and survival of animals.