Irreversible formation of pseudohyphae by haploid

Transcription

1 FEMS Microbiology Letters 119 (1994) Federation of European Microbiological Societies /93/$07.00 Published by Elsevier 99 FEMSLE Irreversible formation of pseudohyphae by haploid Saccharomyces cerevisiae J. Richard Dickinson * School of Pure and Applied Biology, University of Wales College of Cardiff, PO Box 915, Cardiff, CF1 3TL, UK (Received 1 March 1994; revision received and accepted 18 March 1994) Abstract: Culturing haploid strains of Saccharomyces cerevisiae in liquid minimal medium with 2% ethanol and 2% leucine resulted in the formation of long anucleate pseudohyphae. This occurred only with the combination of ethanol as carbon source and leucine as nitrogen source and was independent of mating type. The transition to a pseudohyphal form observed under these conditions appears to be irreversible. These findings further extend our view of the developmental alternatives in this important model eukaryote. Key words: Saccharomyces cerevisiae; Pseudohyphal development; Leucine metabolism; Ethanol Introduction Saccharomyces cerevisiae is well-known as 'budding yeast' because of its pattern of vegetative growth and proliferation. Controversy and interest were stimulated by the description of the formation of pseudohyphae by diploid strains starved for nitrogen on solid media [1]. Gimeno et al. reasoned that pseudohyphal growth is only expected in diploids because they display polar budding (which can be readily envisaged converting into pseudohyphae,) whereas the axial budding of haploids does not lend itself to such development [1]. However, three elm mutations were identified which cause the constitutive production of pseudohyphae by haploids [2]. ELM1 * Corresponding author. Tel: (0222) ; Fax: (0222) (which encodes a putative protein kinase) is involved with the other two ELM genes in suppression of response to nitrogen starvation. Pseudohyphal growth in haploids is said to be rapidly reversible [3]. During the course of investigating the metabolic pathways required for the catabolism of leucine in S. cerevisiae it became necessary to culture the cells in a liquid minimal medium containing leucine and ethanol [Dickinson, unpublished]. The formation of pseudohyphae observed under these conditions is the subject of this report. These results further extend our view of the developmental alternatives in this important model eukaryote. Materials and Methods Prototrophic haploid strain IWD72 (MAT~) which has been described previously [4,5] was SSDI (94)

2 100 used for most experiments. Strain JRD657 (MATa adel his4 leu2 ura3) was also used. Starter cultures were grown in YEPD medium as described before [4,5] to an OD600n m of 10. An inoculum (1 ml) was then removed and cultured in a gyrorotatory incubator (180 oscillations per min) at 30 C in a conical flask filled to 40% nominal capacity in a medium comprising 2% (v/v) ethanol, 2% (w/v) L-leucine and 0.16% (w/v) Difco Yeast Nitrogen Base (without amino acids and ammonium sulphate). Staining with 4',6-diamidino-2- phenylindole ('DAPI') was done using a standard protocol [6]. E t- O 0 Q Results and Discussion Haploid yeast form pseudohyphae when cultured in liquid ethanol minimal medium with leucine Culturing a prototrophic haploid yeast in liquid ethanol minimal medium with leucine as sole nitrogen source gave a diauxic increase in biomass. After a period of proliferation, cell density remained constant for about 100 h before subsequently increasing to reach stationary phase by about 360 h (Fig. 1.) During the prolonged 'diauxic lag' the yeast cells produced pseudohyphae resulting in many bizarre shapes (Fig. 2). These were very long (32-37/~m) i.e. about sixtimes the length of a normal 'mother' cell. This morphological change occurred only with leucine as nitrogen source and ethanol as carbon source. It did not occur with either of the other branched-chain amino acids (isoleucine or valine) or with any other carbon source tested (glucose, fructose, galactose, acetate and glycerol). Although the formation of pseudohyphae occurred in ethanol minimal medium with leucine during the diauxic lag, two results suggest this is not necessary for the formation of pseudohyphae in liquid medium. Firstly, long diauxic lags were observed when cells were cultured in minimal ethanol medium with isoleucine or in 3% (v/v) glycerol minimal medium with leucine, but pseudohyphae were not formed in either case. Secondly, if cells were removed from ethanol minimal medium with leucine at around 330 h (i.e. in deceleration phase, but before final stationary I I I I I I 0 1 O Time (h) Fig. 1. Proliferation in ethanol minimal medium with leucine. Prototrophic haploid IWD72 was cultured in a minimal medium containing 2% (v/v) ethanol and 2% (w/v) L-leucine as described in 'Materials and Methods'. A typical experiment is shown. phase arrest), and inoculated into flesh ethanol minimal medium with leucine, there was no diauxic lag but pseudohyphae were still produced. In the latter case the necessary metabolic adaptations would already have been made. Strain IWD72 used for the experiments decribed above was chosen because it was a prototroph to avoid any possible complications due to the addition of amino acid (or other nitrogenous compound) auxotrophic requirements in minimal medium. This left the possibility that the results were simply a peculiar strain-dependent phenomenon. However, this can be refuted because unrelated strains behaved quite similarly, for example, pseudohyphae were formed by the

3 101 multiply auxotrophic haploid JRD657 (MATa adel- l O0 his4-5191eu2-3,2-112 ura3-5 2 ) which had adenine, histidine and uracil each supplied at 20 /zg ml-1 (there was no need to provide additional leucine, this was the major nitrogen source). The mating type of the strain was irrelevant: both a and a haploids formed pseudohyphae. The transition to a pseudomycelial form is irreversible The irreversibility of pseudomycelial formation was first noted when an aliquot was removed from a culture of IWD72 in ethanol minimal medium with leucine at 357 h and transferred to a fresh batch of liquid YEPD medium to yield an OD600n m of The inoculum contained 24% weird pseudohyphal clumps. The culture was followed until the optical density reached 7.68 (i.e., 8 doublings) by which time the proportion of weird shapes was only 0.1%. The mathematics of cellular proliferation suggested that the yeast-like organisms had proliferated with normal kinetics and the number of pseudohyphae had remained the same (i.e., that the pseudohyphae had not reproduced at all). This has been repeated and the conclusion confirmed by subsequent analysis of micromanipulated individuals onto solid YEPD medium. Also, when cells were stained with DAPI during diauxic lag (120 h after inoculation) in ethanol minimal medium containing leucine, nu- I / / i!. I t i i I Fig. 2. Morphology of haploid Saccharomyces cerevisiae during culture in ethanol minimal medium containing leucine. Prototrophic haploid strain IWD72 was photographed during diauxic lag in ethanol minimal medium containing ieucine. Typical examples are shown. Note the many different morphologies including bent (a), scalloped (b, e), straight (j), cross (h) and branched (f). Often more than one feature was combined. Despite e.g., 'b' and 'j' appearing superficially very different, all these structures had a single main cytoplasm. Separating cell walls and nuclei were confined to the few true proximal cells (see also Fig. 3). The marker bar denotes 60 #.m.

4 102 clei were seen only in the proximal 'cells'. Fluorescence was almost totally absent in the extended pseudohyphal portions, showing that the pseudohyphae did not contain nuclei (Fig. 3.) These results are important for several reasons. Firstly, this is the first description of the production of pseudohyphae by wild-type S. cerevisiae in a liquid medium rather than on the surface of agar as described previously [1-3]. The formation of pseudohyphae in liquid medium had been described earlier in elm mutants [2], but not in wild-type strains. The protocol described here allows experimental conditions to be controlled more precisely and the preparation of bulk quantities of materials for biochemical analysis which was not realistically possible in the systems just mentioned. Also, it does not require special mutant strains. Secondly, the present results show a response which is specific to a single amino acid rather than general nitrogen starvation. Thirdly, the morphological change is irreversible. This requires us to extend our concept of yeast developmental alternatives to include the formation of pseudohyphae without nuclei and which are thus non-reproductive structures (Fig. 4). Fourthly, the changes described are reminiscent of the dimorphism displayed by Candida albicans. It has never been clear whether the morphological transition in C. albicans is of any relevance to its pathogenicity [7,8], but, whilst clearly not identi- t! Fig. 3. DAPI staining of haploid yeast in ethanol minimal medium containing leucine. 120 h after inoculation DAPI staining was done as described elsewhere [6]. Notice that DAPI-staining material could be seen only in the proximal 'cells' and that there was virtually no fluorescence in the extended pseudohyphal portion. The marker bar denotes 50 /Am. mating C v,a diploid and sporulation pseudohypha (e) vegetative cell cycle (3 stationary phase arrest Fig. 4. The developmental alternatives available to haploid Saccharomyces cerevisiae. cal to the yeast-hyphal switch in C. albicans (because it appears to be a 'dead end' in S. cerevisiae), nevertheless, the facility with which a switch can be effected by this cultural technique provides a partial model of the C. albicans dimorphism in the genetically-tractable S. cerevisiae. A number of questions remain unanswered. The first is: 'Why do some, but not all, of the cells in a culture produce pseudohyphae?' Ongoing experiments are designed to test whether conditions c~ be adjusted to increase the proportion of cells which produce pseudohyphae, whether cell cycle stage or cell 'age' are important and if conditional mutants can be obtained. The second question concerns the identity of the molecule which triggers this particular developmental change. Since it occurs only in the presence of leucine, one might guess it is an intermediate or product of leucine catabolism. A more subtle explanation is that an amino acid imbalance has been elicited, an effect easily achieved with leucine in the absence of isoleucine and valine [9]. This could happen because the provision of leucine reduced the biosynthesis and/or utilization of isoleucine and valine. Hence the signal for this newly-described morphology might arise from an abnormal concentration of an intermediate in isoleucine or valine metabolism.

5 103 References 1 Gimeno, C.J., Ljungdahl, P.O., Styles, C.A. and Fink, G.R. (1992) Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68, Blacketer, M.J., Koehler, C.M., Coats, S.G., Myers, A.M. and Madaule, P. (1993) Regulation of dimorphism in Saccharomyces cerevisiae: involvement of the novel protein kinase homolog Elmlp and protein phosphatase 2A. Molec. Cell. Biol. 13, Wright, R.M., Repine, T. and Repine, J.E. (1993) Reversible pseudohyphal growth in haploid Saccharomyces cerevisiae is an aerobic process. Curr. Genet. 23, Dickinson, J.R. and Dawes, I.W. (1992) The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in Saccharomyces cerevisiae. J. Gen. Microbiol. 138, Dickinson, J.R. and Norte, V. (1993) A study of branchedchain amino acid aminotransferase and isolation of mutations affecting the catabolism of branched-chain amino acids in Saccharomyces cerevisiae. FEBS Lett. 326, Sherman, F., Fink, G.R. and Hicks, J.B. (1986) Laboratory Course Manual for Methods in Yeast Genetics. 186 pp. Cold Spring Harbor Laboratory, Cold Spring Harbor. 7 Soil, D.R. (1986) The regulation of cellular differentiation in the dimorphic yeast Candida albicans. BioEssays 5, Odds, F.C. (1985) Morphogenesis in Candida albicans. CRC Crit. Rev. Microbiol. 12, Hinnebusch, A.G. (1992) General and pathway-specific regulatory mechanisms controlling the synthesis of amino acid biosynthetic enzymes in Saccharornyces cerevisiae. In: The Molecular and Cellular Biology of the yeast Saccharomyces Vol. 2 Gene Expression (Jones, E.W., Pringle, J.R. and Broach, J.R. Eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, pp