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Journal of Bacteriology, February 2009, p. 1230-1238, Vol. 191, No. 4
0021-9193/09/$08.00+0     doi:10.1128/JB.01108-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

L. Soledad Guidolin,{ddagger} Andrés E. Ciocchini,{ddagger} Nora Iñón de Iannino, and Rodolfo A. Ugalde*

Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Buenos Aires, Argentina

Received 7 August 2008/ Accepted 30 November 2008

Cyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that have been shown to play an important role in several symbiotic and pathogenic relationships. Cyclic β-1,2-glucan synthase (Cgs), the enzyme responsible for the synthesis of CβG, is an integral membrane polyfunctional protein that catalyzes the four enzymatic activities (initiation, elongation, phosphorolysis, and cyclization) required for the synthesis of CβG. Recently, we have identified the glycosyltransferase and the β-1,2-glucooligosaccharide phosphorylase domains of Brucella abortus Cgs. In this study, we performed large-scale linker-scanning mutagenesis to gain further insight into the functional domains of Cgs. This analysis allowed us to construct a functional map of the enzyme and led to the identification of the minimal region required for the catalysis of initiation and elongation reactions. In addition, we identified the Cgs region (residues 991 to 1544) as being the protein domain required for cyclization and demonstrated that upon cyclization and releasing of the CβG, one or more glucose residues remain attached to the protein intermediate that serves as a primer for the next round of CβG synthesis. Finally, our results indicate that the overall control of the degree of polymerization of CβG is the result of a balance between elongation, phosphorolysis, and cyclization reactions.

* Corresponding author. Mailing address: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, Instituto Nacional de Tecnología Industrial, Edificio 24, Avenida General Paz 5445, San Martín 1650, Buenos Aires, Argentina. Phone: (54-11) 4580-7255. Fax: (54-11) 4752-9639. E-mail: rugalde{at}iib.unsam.edu.ar

{triangledown} Published ahead of print on 12 December 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} L.S.G. and A.E.C. contributed equally to this work.

Journal of Bacteriology, February 2009, p. 1230-1238, Vol. 191, No. 4
0021-9193/09/$08.00+0     doi:10.1128/JB.01108-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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