Membrane Topology Analysis of Cyclic Glucan Synthase, a virulence determinant of Brucella abortus

ANDRÉS E. CIOCCHINI, MARA S. ROSET, NORA IÑÓN DE IANNINO AND RODOLFO A. UGALDE

JOURNAL OF BACTERIOLOGY

American Society for Microbiology

Lugar: Washington, USA; Año: 2004 vol. 186 p. 7205 - 7213

0021-9193

Brucella abortus cyclic glucan synthase (Cgs) is an integral inner membrane protein of 316 kDa (2,831 amino acid residues) responsible for the synthesis of cyclic b-1,2-glucan by a novel mechanism in which the enzyme itself acts as protein intermediate. Cgs uses UDP-glucose as sugar-donor and has the three enzymatic activities necessary for the synthesis of the cyclic polysaccharide  (ie: initiation, elongation and cyclization). Cyclic glucan is required in Brucella abortus for effective host interaction and full expression of virulence. To gain further insight into the structure and mechanism of action of Cgs we studied the membrane topology of the protein using a combination of in silico predictions, a genetic approach involving the construction of fusions between the cgs gene and the genes encoding alkaline phosphatase (phoA) and b-galactosidase (lacZ) and site-directed chemical labeling of lysine residues. We found that Ba-Cgs is a polytopic membrane protein with the amino and carboxyl termini located in the cytoplasm and with six transmembrane segments; transmembrane segment I (residues 419 to 441), II (residues 452 to 474), III (residues 819 to 841), IV (residues 847 to 869), V (residues 939 to 961) and VI (residues 968 to 990). The six transmembrane segments determine four large cytoplasmic domains and three very small periplasmic regions.