FLOCCULATION OF Kloeckera apiculata mc1 INDUCED BY GLUCOSE.

SOSA O. A; MANCA DE NADRA M. C.; FARÍAS M. E.

Orlando, Estados Unidos

Congreso; 106th GENERAL MEETING ASM; 2006

ASM

Flocculation is described as a process characterized by reversible and spontaneous aggregation of yeast cells forming clumps that easily separate from the medium.  Kloeckera apiculata mc1 isolated from wine posses a flocculent phenotype characterized by protein-carbohydrate interaction, stabilized by Ca2+. In previous work we determined that glucose was a stable activator of the flocculent phenotype of Kloeckera apiculata mc1. This carbohydrate can lead to different transduction pathways in yeast. One of these pathways involves protein kinase A (PKA). The PKA could be activated following a cAMP-dependent or cAMP-independent process. The purpose of this study was to determine the role of cAMP on the glucose signal leading to flocculation. Flocculation measurements were performed agitating strongly the yeast suspension and measuring the optical density at 620 nm (OD1). After 15 min at room temperature, the optical density was again measured (OD2). The flocculation percentage was calculated according to the expression: % Floc = ((OD1-OD2)/OD1) x 100. K. apiculata cultured in basal medium (0.2% glucose, 1% peptone, 0.25% malt extract), led to % floc of 42 at 8h incubation at 28ºC. At this time, the addition of 2 and 5% glucose increased the % floc to 59 and 62, respectively. When the basal medium was supplemented with 0.05 mmol/l and 0.1 mmol/l cAMP instead glucose, the flocculation percentage was 124 fold higher than the control. The increase in cAMP could result from intracellular acidification. The protonophore 2,4-dinitrophenol (2,4-DNP)  that produce an intracellular acidification in a non-flocculent yeast culture also conduce to a  flocculation induction.  At 10 mmol/l of 2,4-DNP the flocculation percentage was 53 fold higher than the control. The results suggest that glucose induce the flocculation of Kloeckera apiculata mc1 via an increase of cAMP level in the intracellular medium as consequence of the cytoplasm acidification.