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Biochemistry |
Instituto de Investigaciones Biotecnólogicas, Universidad Nacional de General San Martín, Av. General Paz entre Constituyentes y Albarellos, PO Box 30, (1650) Buenos Aires, Argentina1
Author for correspondence: Nora Iñón de Iannino. Tel: +54 11 4 580 7285. Fax: +54 11 4 752 9639. e-mail: norai{at}inti.gov.ar
In contrast to what happens in Agrobacterium tumefaciens and Rhizobium meliloti, synthesis of periplasmic cyclic 1,2-ß-glucan in Brucella spp. was not inhibited when bacteria were grown in media of high osmolarity. Studies performed with crude membrane preparations showed that cyclic 1,2-ß-glucan synthetase of Brucella spp. was not inhibited by 0·5 M KCl or potassium glutamate; concentrations that completely inhibit the osmosensitive enzymes of A. tumefaciens A348 or R. meliloti 102F34, respectively encoded by the chvB or ndvB genes. The Brucella abortus cyclic 1,2-ß-glucan synthetase gene (cgs) was introduced into A. tumefaciens A1011 chvB and R. meliloti GRT21s ndvB mutants. Synthesis of cyclic 1,2-ß-glucan by the recombinant strains was not inhibited when grown in media of high osmolarity (0·25 M NaCl or 0·5 M mannitol). On the other hand, when the A. tumefaciens cyclic 1,2-ß-glucan synthetase gene was introduced into the R. meliloti GRT21s ndvB mutant, the recombinant strain displayed marked inhibition of cyclic 1,2-ß-glucan synthesis when grown in high-osmolarity media. However, the same gene introduced into a B. abortus cgs mutant background resulted in no inhibition of glucan synthesis at high osmolarity. In vitro studies with crude membranes isolated from recombinant strains revealed that Brucella cyclic 1,2-ß-glucan synthetase was not inhibited by high concentrations of KCl or potassium glutamate even when expressed in Agrobacterium or Rhizobium backgrounds. It was concluded that the lack of effect of high osmolarity on 1,2-ß-glucan synthesis in Brucella is due to two convergent mechanisms: a) the presence of a cyclic 1,2-ß-glucan synthetase that is not affected by concentrations of solutes such as KCl or potassium glutamate and b) either the possible accumulation of compatible solutes that might protect the enzyme from the inhibition by potassium glutamate or the accumulation of other osmolytes that do not affect the 1,2-ß-glucan synthetase.
Keywords: cyclic 1,2-ß-glucan, osmoregulation, Brucella, Agrobacterium
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