Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic beta (1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants

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Journal of Bacteriology, September 1998, p. 4392-4400, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic $beta$ (1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants

Nora Iñón de Iannino,¹ Gabriel Briones,¹^,² Marcelo Tolmasky,³ and Rodolfo A. Ugalde¹^,^*

Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, San Martín 1650,¹ and Comisión Nacional de Energía Atómica, División Agropecuaria, Centro Atómico, Ezeiza 1804,² Argentina, and Institute of Molecular Biology and Nutrition, Department of Biological Science, California State University, Fullerton, California 92834-6850³

Received 25 February 1998/Accepted 25 June 1998

The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB)mutant and an Agrobacterium tumefaciens chromosomal virulence(chvB) mutant. The complemented strains recovered the synthesisof cyclic $beta$ (1-2) glucan, motility, virulence in A. tumefaciens,and nitrogen fixation in R. meliloti; all traits were strictlyassociated with the presence of an active cyclic $beta$ (1-2) glucansynthetase protein in the membranes. Nucleotide sequencing revealedthe presence in B. abortus of an 8.49-kb open reading frame codingfor a predicted membrane protein of 2,831 amino acids (316.2 kDa)and with 51% identity to R. meliloti NdvB. Four regions of theB. abortus protein spanning amino acids 520 to 800, 1025 to 1124,1284 to 1526, and 2400 to 2660 displayed similarities of higherthan 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed thatthe C-terminal 825 amino acids of the Brucella protein, althoughhighly conserved in Rhizobium, are not necessary for cyclic $beta$ (1-2)glucan synthesis. Confirmation of the identity of this proteinas B. abortus cyclic $beta$ (1-2) glucan synthetase was done by theconstruction of a B. abortus Tn3-HoHo1 insertion mutant that doesnot form cyclic $beta$ (1-2) glucan and lacks the 316.2-kDa membraneprotein. The recovery of this mutant from the spleens of inoculatedmice was decreased by 3 orders of magnitude compared with thatof the parental strain; this result suggests that cyclic $beta$ (1-2)glucan may be a virulence factor in Brucella infection.

^* Corresponding author. Mailing address: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de Gral San Martín,Av. General Paz entre Av. Constituyentes y Albarellos, San Martin1650, Pcia de Buenos Aires, Argentina. Phone: 54-1-752-0021. Fax:54-1-752-9639. E-mail: rugalde{at}inti.gov.ar.

Journal of Bacteriology, September 1998, p. 4392-4400, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic (1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants

Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic $beta$ (1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants