INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Microorganisms either secrete or excrete a wide variety of compounds. Some of these substances result from normal metabolic processes, such as alcohols in fermentative yeast. Other compounds are used to promote survival by coordinating development or differentiation (e.g., cAMP-directed chemotaxis in the slime mold Dictyostelium discoideum), by damaging other organisms (e.g., the antifungal, antibiotic, and immunosuppressive drugs FK-506 and rapamycin, produced by species of Streptomyces), or by altering the growth environment more directly (e.g., secreted proteases in Candida albicans, which promote tissue damage and dispersion of infection). Many of these substances are produced in response to external stimuli: nutrient deprivation stimulates cAMP release in D. discoideum and pheromone production in many fungi, whereas host signals stimulate protease secretion in C. albicans. The pathways that recognize such signals are of obvious interest but are poorly characterized.

A better understanding of these pathways in fungi is critical, given that morphological differentiation has been correlated with pathogenicity in many species, most conclusively in C. albicans, in which it has been shown that mutants unable to filament are avirulent (Lo et al., 1997). Conjugation of compatible cell types in the corn pathogen Ustilago maydis results in a filamentous growth form; only this form is virulent, and mutations that block mating differentiation also abrogate pathogenicity (reviewed by Banuett, 1995). Similarly, host invasion in the rice blast fungus Magnaporthe grisea is dependent on the formation of a structure known as an appresorium. Several mutations have been isolated that block this morphological differentiation; these mutations all confer an avirulent phenotype (Mitchell and Dean, 1995; Xu and Hamer, 1996). Thus, a further understanding of the signals that stimulate fungal differentiation, and of the pathways that respond to these signals, may aid in the development of strategies to combat fungal diseases in both animals and plants.

The budding yeast Saccharomyces cerevisiae has a morphological differentiation pathway similar in both structure and regulation to those mentioned above. This phenomenonpseudohyphal, or filamentous, growthis stimulated in diploid cells upon nitrogen starvation. Pseudohyphal cells have an elongated morphology, an altered cell cycle and budding pattern, and enhanced substrate invasion (Gimeno et al., 1992; Kron et al., 1994). The regulation of pseudohyphal differentiation is complex, involving at least two partially interconnected pathways, the mating MAPK pathway and a receptor/G protein/cAMP signaling pathway (Liu et al., 1993; Cook et al., 1997; Kübler et al., 1997; Lorenz and Heitman, 1997; Lorenz et al., 2000). The earliest signaling events are not well understood, but the requirement for two cell-surface proteins, the ammonium permease MEP2 and the G protein-coupled receptor GPR1, implicates the existence of extracellular signals (Lorenz and Heitman, 1998a; Lorenz et al., 2000).

Nitrogen starvation is not the only stimulus that promotes filamentous growth. Poorly used carbon sources such as the starch amylopectin have been reported to induce filamentous growth (Lambrechts et al., 1996). A related phenomenon, haploid invasive growth, allows haploid strains to penetrate the agar substrate in rich medium. As in filamentous growth, cells become elongated and alter their budding pattern, processes that require the same elements of the MAPK cascade that regulate pseudohyphal differentiation (Roberts and Fink, 1994).

Several alcohols can also induce morphological abnormalities. Although ethanol is the primary fermentation product in S. cerevisiae, yeast produces a wide variety of other alcohols, mostly products of amino acid metabolism known collectively as fusel alcohols. This class of alcohols includes compounds such as isoamyl and isobutyl alcohols that are produced particularly under conditions of nitrogen starvation. Dickinson (1994, 1996) showed that several fusel alcohols can promote an aberrant, elongated morphology in S. cerevisiae. In nitrogen-poor conditions, leucine, the precursor of isoamyl alcohol, can also induce elongated cells (Dickinson, 1994).

The morphology of cells growing in the pseudohyphal form is very similar to that of cells exposed to these fusel alcohols. We have further characterized the connection between these alcohol-induced morphological changes and pseudohyphal growth. Several alcohols, notably isoamyl alcohol and butanol, promote a filamentous growth form on solid medium and an elongated and filamentous form in liquid medium. Strikingly, the most dramatic effects are seen in haploid cells in which filamentation is normally repressed. In addition, ethanol, the most prominent alcohol produced by yeast, also enhances filamentous growth in diploid cells. In both of these cases, mutations in the pheromone-responsive MAPK pathway known to block pseudohyphal growth also abrogate alcohol-induced filamentation, indicating a functional link between the nitrogen starvation and alcohol-induced phenomena. These findings suggest that S. cerevisiae has co-opted its own metabolic by-products for use as a signaling mechanism to regulate its development under starvation conditions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Yeast Strains, Plasmids, and Media

Yeast medium and molecular and genetic methods were as described by Guthrie and Fink (1991). Nitrogen-limiting (SLAD) medium was prepared as described previously (Gimeno et al., 1992; Lorenz and Heitman, 1997). Alcohols were added to agar-containing medium after cooling to a concentration of 1% (vol/vol). It should be noted that because of the volatility of the alcohols, the effective concentration in medium is certainly <1%. Butanol was used in the majority of experiments because it induced abundant filamentation while maintaining a low toxicity to the investigator preparing the medium. It was important to incubate plates containing butanol in air-tight bags or enclosed in parafilm separate from control plates, because butanol induced filamentation even on plates lacking the alcohol when in close proximity, presumably as a result of its volatility.

Yeast strains are described in Table 1. Strains SCY40-SCY46 were constructed through a PCR-mediated disruption method with the use of the G418 resistance cassette (Wach et al., 1994) in haploid strains MLY40 and MLY41. The oligonucleotide primers used to construct these disruptions are listed in Table 2. Briefly, the 5' and 3' disruption oligonucleotides, designed to precisely delete the ORF, were used to prime a PCR reaction with the use of plasmid pFA6-KanMX2 (Wach et al., 1994) as a template. PCR products were used to transform haploid strains to G418 resistance. Candidate disruptants were confirmed via PCR with the use of one primer in the 3' flanking region (outside the ORF) and the 5' disruption oligonucleotide (see Table 2). In correct disruptants, this PCR produces a product, whereas in inaccurate integrants it does not. Diploid strains homozygous for these deletions were constructed via a cross between independently derived MATa and MAT haploid deletion strains. YEplac195 (2µURA3; Gietz and Sugino, 1988) was used to complement the Ura auxotrophy of these strains in the majority of experiments. The reporter plasmids pJB207 (pFUS1-lacZ LEU2 CEN) and pIL30-LEU2 (FRE-lacZ LEU2 CEN) have been described (Trueheart et al., 1987; Laloux et al., 1994; Mösch et al., 1996).

Mutagenesis and Screen for Nonfilamentous Mutants

To find mutants insensitive to butanol, we used a transposon-mediated mutagenesis system (Burns et al., 1994). The transposon-tagged libraries were used to transform strains MLY42 (MAT) and MLY43 (MATa) carrying a YEplac195 plasmid. Leu⁺ prototrophs were selected on solid minimal medium plus glucose (YNB medium), pooled, and replated to SLAD + 1% butanol at ~1000 cells per plate. Colonies were screened microscopically after 2-4 d for a nonfilamentous morphology. The insertion points of the transposon were identified as described by Burns et al. (1994). Briefly, a bacterial origin of replication and an ampicillin resistance gene were introduced into transposon sequences by integrating plasmid pRSQ2 into each mutant. Genomic DNA was isolated and cleaved with either EcoRI or HindIII, ligated, and used to transform Escherichia coli strain DH5 to ampicillin resistance. The only productive ligation products will contain the replication origin and Amp^R inserted into the transposon. These plasmids were sequenced to identify the flanking yeast DNA.

To ensure that the transposon insertion was genetically linked to the nonfilamentous phenotype, we crossed the insertion strains to the parent strains and sporulated and dissected the resulting diploid. For reasons that are not clear, spore viability was quite poor in these diploids, although this lethality was not linked to the transposon insertion (the LEU2 marker that tags the insertion segregated independently of the spore lethality phenotype). For mutants for which it was difficult to demonstrate linkage via crosses, the candidate gene was disrupted directly with the use of the G418/PCR approach (see above) and analyzed to ensure that the phenotypes of the disruption strains agreed with those of the original insertion mutations.

Photomicroscopy

Whole colony photographs were taken directly on agar plates with a Zeiss (Thornwood, NY) microscope fitted with a 35-mm Nikon (Garden City, NY) camera. Unless indicated otherwise, whole colonies were photographed at ×25 magnification. Single-cell pictures were taken with a Nikon Eclipse E800 microscope. Images were captured electronically with the use of a MicroMax digital processor (Princeton Instruments) and OpenLab 2.0.3 software (Improvision).

Phenotypic Assays

Budding Pattern Analysis. To assay bud pattern after exposure to butanol, strains were incubated on YPD with or without 1% butanol for 2 d at 30°C. Strains were then restreaked to the same medium and incubated for two doublings (~3-4 h on YPD, 5-6 h on YPD + 1% butanol). Microcolonies were assayed microscopically for either a compact, axial budding morphology or an elongated, bipolar morphology.

Reporter Gene Assays. To assess signaling stimulated by alcohols, Ura Leu strains MLY43 (wild-type MATa), MLY61 (wild-type MATa/), MLY216a (ste12 MATa), and MLY216a/ (ste12/ste12 MATa/) were transformed with the URA3 vector YEplac195 and vectors containing the FUS1-lacZ (pJB207) or FRE-lacZ (pIL30-LEU2) reporters. Strains were grown in YNB containing 1% (vol/vol) butanol or 5 µg/ml -factor for either 4 or 24 h at 30°C. -Galactosidase activity was determined with the use of chlorophenol red--galactopyranoside as a substrate, as described (Cardenas et al., 1994).

Haploid Invasion Assays. The ability of haploid strains to invade the agar substrate was determined as described previously (Roberts and Fink, 1994; Cook et al., 1997). Strains were patched to YPD and incubated for 5 d at 30°C. Surface cells were gently washed off, and the plate was incubated for an additional 24 h at 30°C.

Mating Assays. Qualitative mating assays were performed with the use of strains MLY42 + YEplac181 (ura3-52 leu2::hisG MAT + LEU2-2µ) and MLY43 + YEplac195 (ura3-52 leu2::hisG MATa + URA3-2µ). These strains were incubated together in a patch on YPD with or without 1% (vol/vol) butanol overnight at 30°C and then replica plated to YNB to select for diploids.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Haploid Filamentation Induced by Various Alcohols

Several "fusel" alcohols, such as isoamyl alcohol, n-amyl alcohol, and isobutanol, which result from amino acid metabolism, can induce morphological abnormalities in liquid cultures of S. cerevisiae (Dickinson, 1994, 1996). Moreover, high levels of leucine, a metabolic precursor of isoamyl alcohol, also induce similar hyphae-like extensions (Dickinson, 1994). Given the morphological similarities between cell shape after alcohol exposure and during filamentous, or pseudohyphal, growth, we further analyzed the effects of various alcohols on the growth form of yeast.

Figure 1 shows the effects of a panel of alcohols on colony morphology on nitrogen-limiting (SLAD) medium. As can be seen, this medium allows diploid cells of the 1278b strain background to undergo pseudohyphal differentiation, whereas haploids continue to grow in the yeast form into smooth, round colonies. Several of these alcohols, notably 1-butanol, isobutanol, isoamyl alcohol, and tert-amyl alcohol (see the diagrams in Figure 1 for structures of the more complex alcohols), induced haploid cells to form filamentous colonies when present at 1% (vol/vol) on solid SLAD medium. In Figure 1, we present results with MATa cells; MAT cells filament to a similar degree under these conditions (our unpublished results). Because this differentiation program is normally active only in diploid cells (see the SLAD-only panel in Figure 1) and is repressed in haploid cells, we were surprised to find such vigorous filamentation in haploid cells. These alcohols have little effect on colony morphology of the diploid cell type.

View larger version (73K): [in this window] [in a new window]   Figure 1.   Several alcohols induce haploid filamentation. Wild-type strains MLY41 (MATa) and MLY61 (MATa/) were incubated on solid SLAD medium containing 1% (vol/vol) of the indicated alcohol. The strains were grown at 30°C for 4 d on parafilm-seated plates before being photographed at ×25 magnification. The structures of the less common alcohols are shown below the panels.

The other notable observation from the experiment shown in Figure 1 is the effect of ethanol on colony morphology. Although haploid strains were unaffected, diploid cells incubated on SLAD + 1% (vol/vol) ethanol showed a stimulation of filamentous growth compared with cells grown in alcohol-free conditions. This observation will be examined further at the end of this section.

Filamentous Growth in Liquid Medium

One complication of the analysis of pseudohyphal differentiation is that it occurs only on solid medium. Dickinson (1996) reported that isoamyl alcohol, in particular, would induce morphological abnormalities, described as "hyphal-like extensions," in liquid rich (YPD) medium. We investigated whether these structures were similar in form or regulation to pseudohyphal differentiation.

The time course shown in Figure 2 demonstrates that, at least in this strain background (1278b; in contrast, Dickinson [1996] used the IWP72 background), the phrase "hyphal-like extensions" does not do justice to the morphology of cells grown in liquid YPD + 1% butanol. By 8 h after inoculation into butanol-containing medium, elongated cells can be seen in filamentous clusters. By 30 h, elaborate asters of connected cells have developed. These asters show a remarkable similarity to microcolonies of cells growing in a filamentous form. Although these asters are highly flocculent and sediment readily, they form in liquid medium in rolling cultures. These changes are apparent in both diploids and haploids, but they are far more striking in the haploid cell type. Figure 2 shows results with butanol, although isoamyl alcohol is also effective at inducing these asters (our unpublished results).

View larger version (86K): [in this window] [in a new window]   Figure 2.   A time course of butanol-induced morphological changes in liquid medium. Strains MLY41 (wild-type MATa), MLY61 (wild-type MATa/), and 27-06 (ste12 MATa) were grown overnight in liquid YPD medium, diluted into 5 ml of YPD with (+) or without () 1% (vol/vol) butanol in 15-ml sealed, screw-cap tubes, and grown at 30°C for the indicated times. Cells were spotted to microscope slides and photographed at ×400 magnification. The images at the 30-h time point are presented at half the magnification of the other panels to show the multicellular asters that develop in the haploid wild-type strain.

The filamentous aster structure shown at the 30-h time point in Figure 2 is by no means rare in these cultures. To assess how quantitative these changes are, we counted the number of distinct cell clusters that contained either 1-19 cells or >20 cells per cluster with the use of haploid strain MLY41 (MATa) after 30 h of growth in YPD + 1% (vol/vol) butanol. At this time, 38.4% of cell clusters contained >20 cells, and all of these large clusters had an elongated, filamentous form, similar to that shown in Figure 2. This indicates that the majority of cells are incorporated into multicellular asters when exposed to butanol for 30 h. In contrast, only 9.6% of cell clusters from YPD-grown cultures have >20 cells; moreover, these clusters take the form of tight clumps of cells typical of flocculent haploid strains. Thus, butanol exposure both increases the size and changes the structure of cell clumps isolated from liquid rich medium.

Several alterations contribute to the formation of these structures, the most obvious being cellular elongation. However, it is also clear that haploid cells exposed to butanol alter their budding pattern. Haploid cells typically bud in an axial pattern, in which they choose the location for a new bud adjacent to previous bud sites, whereas diploid cells bud in a bipolar manner, in which buds can emerge from either end of the ovoid cell. The axial pattern tends to form tight clusters of cells (see Figure 2; wild-type MATa without butanol), and the bipolar pattern forms a more elongated cluster of cells. Bipolar buds in cultures of haploid strains can be seen as early as 4 h after exposure to butanol (e.g., compare MATa with butanol to MATa/ without butanol at the 4-h time point). The extent and regulation of the budding pattern switch is analyzed further below. Finally, the maintenance of these filamentous structures is undoubtedly aided by the highly flocculent nature of the 1278b strain. These asters are difficult to separate by sonication, although they can be micromanipulated apart (our unpublished results). As will be shown below, less flocculent strains also show some of these alterations.

Strains lacking the STE12 transcription factor are deficient in both diploid pseudohyphal differentiation and haploid invasive growth, in addition to being unable to mate (Hartwell, 1980; Liu et al., 1993; Roberts and Fink, 1994). Curiously, in the liquid YPD + butanol assay, ste12 mutants show partial phenotypes (Figure 2). The budding pattern of this haploid strain has clearly switched to the bipolar pattern (see also Table 3), but its cells do not elongate. This pattern is also seen in other ste mutants known to affect filamentous growth (we have tested ste7 and ste11 mutant strains).

Analysis of Butanol-induced Phenotypes

Strain Background The 1278b strain background is commonly used for studies of filamentation because of its vigorous response to nitrogen starvation conditions, and the experiments presented in Figures 1 and 2 used this strain. To address the generality of this phenomenon, we tested strains of the W303 and S288c lineages. Both 1278b and W303 show morphological abnormalities after growth for 12 h in liquid YPD + 1% (vol/vol) butanol. W303 is a less flocculent strain than 1278b and does not form multicellular asters like 1278b, but elongated and misshapen cells can be readily observed (Figure 3A). Dickinson (1996) reported that W303 derivatives formed hyphae-like extensions in response to isoamyl alcohol less readily than the strain background he used regularly (IWP72); although we have not quantitated the frequency of aberrant morphologies in W303, Figures 3 and 4 show some examples of these unconventional morphologies in this strain. In contrast, the cell morphology of S288c is unaffected by butanol (Figure 3A). Even after 30 h of growth in liquid medium, the cellular morphology of S288c-derived strains is indistinguishable between cultures with and without butanol.

View larger version (72K): [in this window] [in a new window]   Figure 3.   Effect of strain background on butanol-induced morphological changes. (A) Haploid strains MLY41 (1278b), 10556-24D (W303), and FY69 (S288c) were incubated in liquid YPD with (+) or without () butanol and analyzed after 12 h for morphological abnormalities, as in Figure 2. (B) The same strains as in A were incubated on solid SLAD medium for 4 d at 30°C, scraped off the agar with a toothpick, and analyzed microscopically, as in Figure 2.

View larger version (121K): [in this window] [in a new window]   Figure 4.   Aberrant morphologies induced in W303 after butanol exposure. Strain 10556-24D, harboring the URA3 vector YEplac195 and the LEU2 vector YEplac181, was incubated on solid SLAD medium with or without 1% butanol for 4 d at 30°C. Cells were scraped off the plate and resuspended in 10 µl of water on a microscope slide. (A) Typical cell morphology of this strain on medium lacking butanol. (B-I) Different morphologies of this W303 strain on SLAD + butanol.

Budding Pattern We assayed the budding pattern of strains grown in the absence or presence of butanol to determine the extent to which haploid strains switch to a bipolar budding pattern and the effect of strain background on this phenomenon. In this assay, strains were incubated on solid YPD with or without 1% butanol for 2 d at 30°C and then restreaked to fresh medium. After 4 h (YPD) or 6 h (YPD + 1% butanol), microcolonies were examined for a tightly clustered (axial) or an elongated (bipolar) pattern. Only microcolonies with four or more cells were counted in this analysis.

Reporter Assays Two reporters have been developed that respond to activation of the STE MAPK pathway. The first uses the promoter of FUS1, encoding a cell surface protein that is dramatically up-regulated by pheromone treatment. A FUS1-lacZ fusion gene is induced several hundredfold during mating response (Trueheart et al., 1987). The other reporter uses the filamentation response element (FRE) found in the control sequences of the Ty1 retrotransposon (a similar element is found in the TEC1 promoter; Madhani and Fink, 1997) and has been correlated to MAPK activation in low-nitrogen conditions that stimulate diploid pseudohyphal growth (Laloux et al., 1994; Mösch et al., 1996; Madhani and Fink, 1997). These reporters are specific, in that the FUS1-lacZ gene does not respond to nitrogen starvation and the FRE-lacZ gene does not respond to pheromone (Mösch et al., 1996; Madhani and Fink, 1997). We grew strains harboring each of these reporter genes in YNB with or without 1% butanol for either 4 or 24 h. At the 24-h time point, the morphology of haploid cells grown in YNB + 1% butanol was similar to the morphology of cells grown in rich (YPD) medium plus butanol, with abundant filamentous asters present. As shown in Figure 5, we found that neither of these reporters is induced by the presence of butanol. Other findings presented here, namely that butanol-treated ste12 cells do not elongate (Figure 2) and do not form filamentous colonies (see Figure 7), clearly imply a role for STE12 and the MAPK pathway in alcohol-induced filamentation. Yet, the lack of reporter activation after butanol treatment suggests that another avenue of specialization exists to regulate STE12 activity based on an upstream input.

View larger version (27K): [in this window] [in a new window]   Figure 5.   Reporter gene activity after butanol exposure. MATa and MATa/ strains, both wild type (MLY41, MLY61) and ste12 (MLY216a, MLY216a/), were transformed with an empty URA3 vector (YEplac195) and a LEU2 vector containing either the FUS1-lacZ reporter (pJB207; top panels) or the FRE-lacZ reporter (pIL30-LEU2; bottom panels). The strains were then grown in YNB medium overnight (in duplicate) and split three ways; to the three cultures were added nothing, 1% (vol/vol) butanol, or 10 µM -factor. -Galactosidase activity was assayed after 4 or 24 h at 30°C in rolling cultures.

Induction of Filamentous Colony Morphology Filamentation has been difficult to detect in rich medium; one explanation for this finding is that the rapid growth rate of cells in rich conditions allows colonies to overgrow and obscure any filaments that are formed. The central role for nitrogen starvation in regulating diploid pseudohyphal differentiation led us to ask whether nitrogen deprivation was also required for the colony phenotype induced by butanol or whether other starvation signals may allow a filamentous colony morphology after exposure to butanol. To test this question, we incubated strains on solid medium containing altered levels of nitrogen or carbon. The baseline in this experiment is YNB, a synthetic minimal medium with high levels of ammonium sulfate as a nitrogen source and glucose as a carbon source (37 mM ammonium, 2% glucose). We then used medium with 50 µM ammonium and 2% glucose (SLAD) or 37 mM ammonium and either 0.2% or 0.02% glucose. As shown in Figure 6, decreasing the glucose concentration also allows filamentous growth (as assayed by colony morphology) in the presence of butanol, suggesting that this alcohol can enable environmental inputs other than nitrogen starvation to trigger this differentiation pathway. Lambrechts et al. (1996) found that poorly used carbon sources such as the starch amylopectin can also induce filamentous growth, but pseudohyphal differentiation has not been observed previously on low-glucose medium. Thus, we conclude that nitrogen starvation, per se, is not required for the haploid, butanol-induced, filamentation phenotype. This phenomenon is also observed after starvation for carbon, and we suggest that it is simply a slow growth rate that is necessary to observe filaments upon butanol treatment.

View larger version (86K): [in this window] [in a new window]   Figure 6.   Butanol stimulates filamentation in glucose-poor medium. The MATa strain MLY41 (top panel) and the MATa/ strain MLY61 (bottom panel) were incubated on YNB (2% glucose, 37 mM ammonium), SLAD (2% glucose, 50 µM ammonium), or medium with 37 mM ammonium and either 0.2% or 0.02% glucose, either with (+) or without () 1% (vol/vol) butanol. Strains were grown for 4 d at 30°C.

Other Phenotypes In addition to the phenotypes described above, we determined whether butanol would affect the processes of haploid invasive growth and mating, both of which require the pheromone-responsive MAPK pathway. The finding that butanol does not affect expression of either the FUS1-lacZ or the FRE-lacZ reporter gene (Figure 5) suggested that butanol would not alter invasive growth or mating, but each of these processes is complex, and reporter activity may not accurately reflect the effects of butanol. We found that butanol neither inhibits nor enhances haploid invasive growth. Haploid strains continue to invade the agar substrate, although invasion is delayed, presumably as a result of the slower growth rates on media containing butanol. Butanol does not allow diploid strains or haploid ste12 mutants to invade the agar substrate (our unpublished results). We also investigated mating in the presence of butanol and found that haploid strains are still able to conjugate in the presence of 1% butanol (our unpublished results). Thus, despite the multiple functions of the MAPK pathway, the cell remains able to distinguish between the specific inputs to properly regulate these differentiation events.

Genetic Analysis of Butanol-induced Filamentation

Analysis of Known Mutations A number of mutations have been identified that block filamentation. Among these are elements of the MAPK pathway, including STE12 and its binding partner TEC1, and upstream elements postulated to be part of the nitrogen sensor, such as the G protein/receptor complex GPA2/GPR1 and the ammonium permease MEP2 (Liu et al., 1993; Gavrias et al., 1996; Lorenz and Heitman, 1997, 1998a; Lorenz et al., 2000). When assayed for butanol-induced phenotypes (Figure 7), ste12 and tec1 mutations block colony filamentation stimulated by butanol. In some colonies of ste12 mutant strains grown in the presence of butanol, a weak residual filamentation can be seen, whereas the nonfilamentous phenotype of tec1 mutant strains is very tight (Figure 7). Differences in the severity of phenotypes conferred by these two mutations have been observed, and we have previously proposed that TEC1 may have STE12-independent functions in the regulation of filamentous growth (Lorenz and Heitman, 1998b). In contrast, butanol still promotes filamentous growth in strains lacking GPA2, GPR1, or MEP2, both on low-nitrogen solid medium (Figure 7) and in liquid rich medium; in fact, in MATa/ cells, butanol suppresses the pseudohyphal growth defect of gpa2/gpa2, gpr1/gpr1, and mep2/mep2 strains on solid SLAD medium (our unpublished results). These findings suggest that this alcohol bypasses the nutrient-sensing apparatus but still requires elements of the MAPK pathway.

View larger version (41K): [in this window] [in a new window]   Figure 7.   Genetic analysis of the butanol-induced phenomenon. Strains MLY41 (wild-type MATa), MLY61 (wild-type MATa/), 27-06 (ste12 MATa), MLY183a (tec1 MAT), MLY132a (gpa2 MATa), MLY232a (gpr1 MATa), and MLY108a (mep2 MATa) were incubated on SLAD medium with or without 1% (vol/vol) butanol. After 4 d at 30°C, colonies were photographed at ×25 magnification.

mep2

gpa2

gpr1

ste20/ste20

Screen for Mutations That Block Alcohol-induced Filamentation We next screened for mutations that inhibit butanol-induced filamentation, with the use of a random, transposon-tagged insertional mutagenesis scheme (Burns et al., 1994). We found 12 transposon-linked mutations that, when rescued from the genome, identified nine genes. A few of these represent genes known to regulate filamentous growth, such as STE12 (Liu et al., 1993), and the polarity establishment gene BUD8 (Mösch and Fink, 1997), mutation of which disrupts the diploid bipolar budding pattern and causes cell wall defects (Lussier et al., 1997). Although BEM1 and BEM4 had not previously been associated with filamentous growth, this finding was not surprising given their known role in polarity establishment. BEM1, in particular, is required for polarized growth both in budding and in mating response and interacts with STE20, the first kinase necessary for both mating and filamentous/invasive growth (Chenevert et al., 1994; Leberer et al., 1996). bem1 mutants have delocalized cortical actin and chitin (Chenevert et al., 1992), and it is likely that BEM1 links the MAPK signaling cascade to changes in the actin cytoskeleton. Similarly, BEM4 interacts with Rho-type GTPases that regulate actin cytoskeletal reorganization (Hirano et al., 1996; Mack et al., 1996).

mep2

flo11

gpa2

mep2

Ethanol-induced Hyperfilamentation

As shown in Figure 1, we identified a role for ethanol in stimulating hyperfilamentation of diploid cells in nitrogen-limiting conditions. As with the butanol-induced phenotypes, we have analyzed the genetic regulation of this phenomenon. Mutations in STE12 and TEC1 blocked ethanol-induced hyperfilamentation (Figure 8), as was also the case with butanol, although weak filamentation could be seen in ste12 mutants (Figure 8). In contrast, ethanol suppressed the pseudohyphal defects of mep2/mep2, gpa2/gpa2, and gpr1/gpr1 strains. Again, these data are similar to our findings for the regulation of butanol-induced filamentation in haploid cells, which is a requirement for STE12/TEC1 signaling, but independence from the MEP2 and GPR1/GPA2 nutrient sensors.

View larger version (39K): [in this window] [in a new window]   Figure 8.   Genetic analysis of the ethanol-induced phenomenon. Strains MLY41 (wild-type MATa), MLY61 (wild-type MATa/), HLY352 (ste12/ste12 MATa/), MLY183a/ (tec1/tec1 MATa/), MLY108a/ (mep2/mep2 MATa/), MLY132a/ (gpa2/gpa2 MATa/), and MLY232a/ (gpr1/gpr1 MATa/) were incubated on SLAD medium with or without 1% (vol/vol) ethanol. After 2 d at 30°C, colonies were photographed at ×25 magnification.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The data presented here demonstrate a role for various alcohols in both cellular and colony morphology in S. cerevisiae. Some of these alcohols are products of amino acid metabolism, such as isoamyl alcohol and butanol, which accumulate specifically in conditions of nitrogen starvation. These alcohols stimulate haploid cells to differentiate into a filamentous form similar to diploid-specific, nitrogen starvation-induced, pseudohyphal development. Both of these phenomena involve an elongated cell morphology, alterations in budding pattern, and a dependence on elements of the pheromone-responsive MAPK pathway.

These phenotypes are strongly dependent on the particular yeast strain under study. The structures formed by strains of the 1278b background are strikingly similar to pseudohyphal cells, with elongated, cylindrical cells. W303 derivatives, however, adopt a variety of morphologies, including ellipsoidal yeast-form cells, elongated, cylindrical shapes, and rounded yeast cells projecting a thin, hyphal projection reminiscent of germ tube formation in C. albicans. In contrast, strains of the S288c lineage seem completely unaffected by the presence of these alcohols.

This phenotype represents the third process that the pheromone-responsive MAPK pathway regulates in haploid cells. First is mating response, which is activated by pheromone binding to a cell surface receptor/G protein complex and signals through the FUS3 MAPK associated with the STE5 scaffolding protein. Second is haploid invasion, a process stimulated by unknown stimuli, which does not require STE5 and signals primarily through the KSS1 MAPK. Third is butanol-induced filamentation, which also uses the same MAPK pathway. We have not, however, analyzed the roles of STE5, FUS3, and KSS1 in detail. Reporter genes responsive to either pheromone activation (FUS1-lacZ) or filamentation/invasion activity (FRE-lacZ) do not respond to butanol, indicating again that there is a specialization of this signaling pathway. Furthermore, the phenotypes of ste20 mutations differ in these assays. In the 1278b background, ste20/ste20 mutant strains have a severe defect in pseudohyphal growthmore severe, in fact, that other ste mutants. However, ste20 haploid strains have only a modest defect in mating and essentially no defect in butanol-induced filamentation (our unpublished observations). Thus, there must be pathway-specific specialization at the STE20 step, as there is for STE12 and the MAPKs KSS1 and FUS3.

Although these morphological changes are at least partially dependent on the STE MAPK pathway, they are independent of elements of the nutrient sensors, including GPA2, GPR1, and MEP2. This suggests that the alcohols are bypassing the need for nitrogen starvation, an idea supported by the behavior of strains grown in rich (YPD) liquid medium plus butanol (Figure 2) and by the filamentous colony morphology on nitrogen-rich, glucose-poor medium (Figure 6). This would be similar in concept to haploid invasive growth, in which haploid cells grown on rich solid medium invade the agar substrate. Because this occurs on rich medium, it is unlikely to be a nutrient response, and the mutations in the nutrient-sensing machinery have either no defect in invasive growth (mep2; Lorenz and Heitman, 1998a) or a severe defect (gpa2 and gpr1; Pan and Heitman, 1999).

A screen for additional mutants that affect butanol-induced filamentous growth identified several genes not previously appreciated to have filamentation phenotypes. Genes such as BEM1, BEM4, BUD8, and FIG1 have been implicated in polarized growth; thus, their involvement in this phenomenon is not surprising. The effects of other genes, such as CHD1, which encodes a transcription factor homologue, are less obvious. With the exception of only the putative mitochondrial helicase HMI1 (and perhaps FIG1), mutations that block butanol-induced (haploid) filamentation also block nitrogen-induced (diploid) filamentation.

In addition to the haploid phenotypes, we found that ethanol induces hyperfilamentation of diploid strains on low-nitrogen medium. Ethanol has no effect on the colony or cellular morphology of haploid cells. Again, this phenotype requires TEC1 and the pheromone-response elements that regulate filamentous growth, but it does not require GPA2, GPR1, and MEP2. Thus, as with the haploid phenotype, ethanol stimulates filamentation in a manner that bypasses the nutrient-sensing machinery.

Why do these alcohols have effects on colony morphology? It has been suggested that pseudohyphal differentiation is a means by which yeast cells scavenge for nutrients in scarce conditions (Gimeno et al., 1992). Because these alcohols bypass the presumptive nutrient-sensing apparatus, it is possible that they represent an alternative means to sense nutrient availability. As yeast cells metabolize the available nutrients and their own proteins and amino acids as nitrogen sources, the concentration of by-products such as isoamyl alcohol and, in particular, ethanol increases. Yeast may have a mechanism to estimate nutrient availability based on the levels of its own by-products. Alternatively, these alcohols may be toxic to the cell, and high concentrations may stimulate filamentous growth to allow the cell to escape the poisoned environment. Indeed, the presence of butanol, isoamyl alcohol, or ethanol (at high concentrations) slows growth rates, supporting this idea.

A third possibility is that yeast uses the concentration of these alcohols as a mechanism to sense population density and coordinate development appropriately. Quorum sensing of this nature is common in bacteria. Population density is one signal that regulates competence development in Bacillus subtilis. A secreted pheromone (ComX) activates a two-component signaling pathway once it passes a threshold concentration (reviewed by Grossman, 1995). Other bacteria, such as Vibrio fischeri, control autofluorescence based on population density with the use of secreted autoinducers, mostly related to N-acyl homoserine lactones (reviewed by Hellingwerf et al., 1998). Quorum-sensing systems have also been described in both plant and human pathogens (Agrobacterium tumefaciens and Pseudomonas aeruginosa, respectively) to regulate expression of virulence factor genes.

Whatever the reason for this phenomenon in yeast, there must be a cellular mechanism to sense the presence of these alcohols. Although the STE MAPK pathway is required for the full expression of butanol-induced phenotypes, this pathway is not likely to represent the only butanol-responsive signaling system in yeast. As shown in Figure 2 and Table 3, haploid ste12 mutants change their budding pattern in response to butanol; thus, this element of the phenotype is necessarily independent of the MAPK pathway. For this reason, we expect there to be a system (or systems) to recognize the presence of these alcohols and transduce a signal to multiple downstream pathways, including the MAPK pathway.

It is possible that, rather than sensing the alcohols themselves, cells sense intermediates in the conversion of amino acids to alcohols. By this model, addition of alcohols to the medium would be expected to increase the concentration of these intermediates in the cell. Several distinct pathways have been identified that convert leucine to isoamyl alcohol or valine to isobutyl alcohol (Dickinson et al., 1997, 1998); the pathways are overlapping and interconnected, supporting the idea that an intermediate could be the relevant signaling molecule. We tend not to support this idea, though, because we also see these affects with 1-butanol and tert-amyl alcohol, which, although related to isoamyl and isobutyl alcohols, are not derived from any common amino acids. Moreover, neither -ketoisocaproic acid (derived from leucine) nor -ketoisovaleric acid (derived from valine) affected colony morphology when added to solid SLAD medium (Lorenz, Cardenas, and Heitman, unpublished observations).

There are a few precedents for the idea of branched chain amino acids or their derivatives in signaling roles. One study has proposed a role for branched chain amino acids (such as leucine and valine) in the control of translation (Xu et al., 1998). Addition of these amino acids to pancreatic -cells stimulated phosphorylation of the PHAS-I and p70^S6k proteins. Phosphorylated forms of both PHAS-I and p70^S6k stimulate translation, PHAS-I via interactions with the mRNA cap-binding protein eIF-4E and p70^S6k via phosphorylation of ribosomal protein S6. Although this study correlated the effects with essential versus nonessential amino acids, it also showed that -ketoisocaproic acid had this effect as well, suggesting that the relevant signaling molecule may be a by-product rather than the amino acids themselves. In microorganisms, branched chain amino acid metabolism has been linked to growth and development in the Gram-negative bacterium Myxococcus xanthus (Toal et al., 1995). Mutations at the esg locus confer growth defects in minimal medium and defects in aggregation and differentiation during development of multicellular fruiting bodies. The esg locus encodes a branched chain keto acid dehydrogenase, an enzyme that converts -ketoacids (the transamination products of branched chain amino acids) to short, branched chain fatty acids. Indeed, several fatty acids can rescue the developmental defects of esg mutants. In this case, the signaling molecule is not an alcohol by-product but a fatty acid; nevertheless, the involvement of branched chain amino acid metabolism is shared between development in M. xanthus and the phenotypes described here for S. cerevisiae.

Butanol-induced filamentous growth offers an additional advantage that has thus far been impossible in the analysis of filamentation. The recent advent of DNA array or chip experiments presents the possibility of understanding the transcriptional program of filamentous growth. This is undoubtedly complex, because a large number of transcriptional regulators (perhaps 18 to date) have been linked with filamentous growth in many laboratories. Standard pseudohyphal growth is confined to solid medium, and not all cells become elongated or invasive, thus making array experiments extremely difficult. Butanol allows "filamentous" growth in liquid medium, and virtually all cells show some aspects of this behavior, making the butanol-induced phenomenon amenable to array analysis. These experiments are under way and hopefully will shed light on the regulation of filamentous growth.