Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Thu, 13 Dec 2018 14:36:19 Microbiology (1 999), 145, 309-3 1 6 Printed in Great Britain Down-regulation of the expression of PKC7 and SRB7lPSA7IVIG9, two genes involved in cell wall integrity in Saccharomyces cerevisiae, causes flocculation Nianshu Zhang, David C. J. Gardner, Stephen G. Oliver and Lubomira I. Stateva Author for correspondence: Lubomira I. Stateva. Tel: $44 161 200 4208. Fax: +44 161 236 0409. e-mail : lubomira.stateva@umist.ac.uk Department of Biomolecular Sciences, UMIST, PO Box 88, Manchester M60 lQD, UK The cell wall integrity determinants PKCl and SRB7IPSAllVIG9 of Saccharomyces cerevisiae were expressed under the control of the tightly regulated promoter pMET3. Substitution of the cell-cycle-regulated SRBlIPSAl native promoter with pMET3 led to faster cell growth, larger cell volumes, and a twofold reduction of the steady-state SRBlIPSAl mRNA level. In addition, the new pattern of expression of SRBlIPSAl resulted in a dominant flocculation phenotype at all phases of batch growth. By contrast, expression of PKCl from pMET3 increased the flocculation capacity of cells only at stationary phase. Methionine-mediated repression of either PSA 7ISRB7 or PKC7 resulted in enhanced cell clumping. Cells in which both these genes had been replaced with their respective pMET3-regulated cassettes were highly flocculent under both expression and repression conditions. These results suggest that greater exposure of flocculin on the cell surface, caused by either cell wall distortion (through depletion of Pkcl p)- or aberrant regulation of mannosylation (through constitutive production of Srblp), results in an increased flocculation ability. Keywords : MET3 promoter, SRBl IPSAI, PKCl, cell wall, yeast flocculation INTRODUCTION The yeast cell wall is a dynamic organelle responsible for a number of cellular functions, the most important being physical and osmotic protection, selective permeability, and cell-cell recognition and adhesion during mating and flocculation (Stratford, 1994). Unlike adhesion in mating, which is induced by highly specific pheromones, flocculation is an asexual aggregation of cells which is a very useful characteristic in industrial yeast strains. Flocculation is exploited in fermentations such as beer- brewing, wine-making, and fuel ethanol production because it leads to efficient separation of cells from the fermentation liquor (Esser & Kues, 1983). Two types of flocculation phenotypes have been de- scribed (Stratford & Assinder, 1991). The Flol type, caused by FLOl /FLOS/FL08, is Ca2+-dependent and Abbreviation : GPI, glycosylphosphatidylinositol. inhibited by mannopyranoses. The NewFlo phenotype, on the other hand, is prevented by both manno- and glucopyranoses. The FLOl gene, which is located on chromosome I, has been reported to encode a glyco- sylphosphatidylinositol (GP1)-anchored cell-surface protein with its amino terminus exposed to the medium (Bony et al., 1997; Straver et al., 1994). FL05 is highly homologous to FLOl (Bidard et al., 1994) and is also found on chromosome I. FL08, previously mapped to chromosome I and said to be allelic to FLOl (Teunissen et al., 1995b), has recently been reassigned to chromo- some V and demonstrated to mediate flocculation via transcriptional activation of FLOl (Kobayashi et al., 1996). More recently, a new flocculation gene, named FL02, has been cloned and localized to chromosome XII; its function remains unclear, although it can complement flo1 mutations (Sieiro et al., 1997). Other genes, such as TUPl and SNN6, also act on yeast cell flocculation via transcriptional regulation (Teunissen et al., 1995a). Flocculation in Saccharomyces cerevisiae is thought to 0002-2851 0 1999 SGM 309