Membrane topology analysis of cyclic glucan synthase, a virulence determinant of Brucella abortus.

Brucella abortus cyclic glucan synthase (Cgs) is a 316-kDa (2,831-amino-acid) integral inner membrane protein that is responsible for the synthesis of cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. B. abortus Cgs uses UDP-glucose as a sugar donor and has the three enzymatic activities necessary for synthesis of the cyclic polysaccharide (i.e., initiation, elongation, and cyclization). Cyclic glucan is required in B. abortus for effective host interaction and complete expression of virulence. To gain further insight into the structure and mechanism of action of B. abortus 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 beta-galactosidase (lacZ), and site-directed chemical labeling of lysine residues. We found that B. abortus Cgs is a polytopic membrane protein with the amino and carboxyl termini located in the cytoplasm and with six transmembrane segments, transmembrane segments 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.