Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis.
Curr Genet (1995) 27:110-122
9 Springer-Verlag 1995
Maria Jos6 Neves - Stefan Hohmann 9 Walter Bell Franqoise Dumortier 9 Kattie Luyten 9 Jos6 Ramos Philip Cobbaert 9 Wire de Koning 9Zoya Kaneva Johan M. Thevelein
Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomycescerevisiaemutants in trehalose biosynthesis Received: 12 July 1994
The GGS1/TPS1 gene of the yeast Saccharomyces cerevisiae encodes the trehalose-6-phosphate syn-
Abstract
thase subunit of the trehalose synthase complex. Mutants defective in GGS1/TPS1 have been isolated repeatedly and they showed variable pleiotropic phenotypes, in particular with respect to trehalose content, ability to grow on fermentable sugars, glucose-induced signaling and sporulation capacity. We have introduced thefdpl, cifl, bypl and glc6 alleles and the ggsI/tpsl deletion into three different wild-type strains, M5, SP1 and W303-1A. This set of strains will aid further studies on the molecular basis of the complex pleiotropic phenotypes of ggsl/tpsl mutants. The phenotypes conferred by specific alleles were clearly dependent on the genetic background and also differed for some of the alleles. Our results show that the lethality caused by single gene deletion in one genetic background can become undetectable in another background. The sporulation defect of ggsl/tpsl diploids was neither due to a deficiency in G 1 arrest, nor to the inability to accumulate trehalose. Ggsl/tpslA mutants were very sensitive to glucose and fructose, even in the presence of a 100-fold higher galactose concentration. Fifty-percent inhibition occurred at concentrations similar to the K m values of glucose and fructose transport. The inhibitory effect of glucose in the presence of a large excess of galactose argues against an overactive glycolytic flux as the cause of the growth defect. Deletion of genes of the glucose carrier family shifted
M. J. Neves. S. Hohmann. W. Bell. F. Dumortier. K. Luyten E Cobbaert 9Z. Kaneva 9J. M. Thevelein ([]) Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit te Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Belgium J. Ramos Departamento de Microbiologia, Escuela Superior de Ingenieros Agronomos, Apartado 3048, Universidad de Cordoba, E-14071 Cordoba, Spain Communicated by F. K. Zimmermann
the 50% growth inhibition to higher sugar concentrations. This finding allows for a novel approach to estimate the relevance of the many putative glucose carrier genes in S. cerevisiae. We also show that the GGS1/TPS1 gene product is not only required for the transition from respirative to fermentative metabolism but continuously during logarithmic growth on glucose, in spite of the absence of trehalose under such conditions. Key words
Yeast - Trehalose synthase
GGS1/TPS1 gene 9 Isogenic background Glucose transport 9 Sporulation
Introduction In the yeast Saccharomyces cerevisiae several mutants, fdpl, cifl, bypl, sstl and glc6, in the GGS1/TPS1 gene have been isolated using different approaches. The fdpl mutant was unable to grow on rapidly fermented sugars, like glucose and fructose, while the cifI mutant was able to grow on glucose but not on fructose. In the presence of such rapidly fermented sugars both mutants showed hyperaccumulation of sugar phosphates and loss of ATE This was thought to be due to the absence of glucose-induced inactivation of fructose-1,6-bisphosphatase (van de Poll et al. 1974; Navon et al. 1979). Another phenotype identified originally in the fdpl mutant was a marked alteration in the ratio of independence (activity in the absence/presence of glucose-6-phosphate) of the glycogen synthase enzyme, although the glycogen level was reported to be unaffected. The connection with the growth defect on glucose was unclear (van de Poll and Schamhart 1977). Subsequently, Panek's group showed that thefdpl and cifl mutants were both allelic to the sstl mutant and like that mutant had very low trehalose-6-phosphate synthase activity and a very low trehalose level (Charlab et al. 1985). The addition of glucose to S. cerevisiae cells growing on non-fermentable carbon sources triggers a wide variety of regulatory effects. We have shown previously that the
111
fdpl mutant is not only deficient in glucose-induced inactivation of fructose-l,6-bisphosphatase, as originally reported by van de Poll et al. (1974), but also in many other glucose-induced regulatory phenomena (Van Aelst et al. 1991, 1993). We have also cloned a suppressor gene, FPS1, which restored growth of thefdpl mutant on glucose, but not the glucose-induced regulatory responses (Van Aelst et al. 1991). From this result we concluded that the defect in glucose-induced signaling was not a side-effect of the metabolic deficiency, but truly reflected involvement of the FDP1 gene product in a general glucose sensing system. Therefore, and because of the mix-up with the FBP1 gene, the structural gene for fructose-l,6-bisphosphatase, we re-named the gene GGS1 (Thevelein 1992; Van Aelst et al. 1993). The bypl mutant was originally isolated as causing a synthetic growth defect on glucose medium to mutants lacking one of the two genes encoding phosphofructokinase (Breitenbach-Schmitt et al. 1984). We showed that the bypl mutant is allelic to fdpl and cifl, displaying a similar deficiency in glucose-induced signaling and a leaky growth deficiency on glucose (Hohmann et al. 1992). The glc6 mutation was isolated in a screen for mutants deficient in glycogen accumulation. The glc6 mutant displays no growth defect on fermentable sugars (Cannon et al. 1994). The GGS1 gene has been cloned independently as complementing the fdpl and bypl mutants (Hohmann et al. 1992; Van Aelst et al. 1993), the cifl mutant (Gonzfilez et al. 1992), and also the glc6 mutant (Cannon et al. 1994). The nature and position of the different mutations has also been determined: fdpl and glc6 are missense mutations at amino-acid positions 64 and 223, respectively, while cifl and bypl are nonsense mutations at amino-acid positions 182 and 317, respectively (Van Aelst et al. 1993; Cannon et al. 1994). Unexpectedly, the same gene was also cloned by two other groups as encoding the smallest subunit of the trehalose synthase complex and called respectively TPS1 (Bell et al. 1992) and TSS1 (Vuorio et al. 1993). Presumably, the GGS1/TPS1 gene encodes the catalytic subunit for trehalose-6-phosphate synthase activity, since its expression in an Escherichia coli otsA mutant, which is defective in trehalose-6-phosphate synthase, restores trehalose synthesis (McDougall et al. 1993). In addition, overexpression of GGS1/TPS1 results in higher trehalose levels (Hohmann et al. 1994; Van Dijck et al., manuscript in preparation). The trehalose synthase complex in yeast comprises both enzymatic activities required to synthesize trehalose: trehalose-6-phosphate synthase and trehalose6-phosphate phosphatase (Vandercammen et al. 1989). It is composed of three subunits, the 56-kDa GGS1/TPS1 gene product, the 103-kDa TPS2 gene product, which is most probably responsible for the phosphatase activity (De Virgilio et al. 1993), and the largest (123 k D a / l l 5 kDa) subunit, which is probably encoded redundantly by two genes, TSL1 (Vuorio et al. 1993) and TPS3 (Manning et al. 1992; Hohmann S., De Virgilio C., Bell W., Vuorio O., Londesborough J., Wiemken A. and Thevelein J. M., unpublished results). The largest subunit is proposed to have
a regulatory function. Deletion of the GGS1/TPS1 gene causes a growth defect on fermentable sugars and absence of glucose-induced signaling (Gonz~lez et al. 1992; Van Aelst et al. 1993). Deletion of TPS2 causes accumulation of trehalose-6-phosphate under conditions where wildtype strains accumulate trehalose: e.g., in stationary-phase cells and during sublethal heat treatment (De Virgilio et al. 1993). How mutations in an enzyme involved in trehalose biosynthesis can lead to such diverse phenotypic effects is puzzling. A complicating aspect in all studies ofpleiotropic yeast mutants is that the different alleles are often obtained in different genetic backgrounds. This is also true for the many ggsl/tpsl alleles that have been characterized. Up to now, in spite of its importance, few systematic studies have been performed on the influence of the genetic background on pleiotropic phenotypes of yeast mutants. In the present study, simply by using a different commonly used wildtype strain, we show that the same allele can either cause a very strong phenotype or have very little effect. Initially, the growth defect of the ggsl/tpsl mutants was thought to be due to lack of glucose-induced inactivation of fructose-l,6-bisphosphatase, hence the name 'fdpl'. This could generate a wasteful ATP-splitting futile cycle at the level ofphosphofructokinase (van de Poll et al. 1974; Navon et al. 1979). However, subsequent experiments showed that such a futile cycle was not operative in these mutants (Bafiuelos and Fraenkel 1982). Alternatively, van de Poll and Schamhart (1977) suggested that thefdpl mutant was deficient in a hypothetical feedback-inhibition system of glycolysis on sugar transport. In S. cerevisiae cells the intracellular level of free glucose is always very low compared to the external level. This appears to indicate that sugar transport is slower than sugar phosphorylation and that the hyperphosphorylation of sugar in the fdpl, cifl and ggsl/tpslA mutants is due to faster glucose transport rather than faster glucose phosphorylation. In spite of this, it has been reported recently that trehalose-6phosphate acts as an inhibitor of hexokinase PII- and to a lesser extent PI-activity in vitro (Blfizquez et al. 1993). In mammalian cells hexokinase is well known to be controlled through feedback-inhibition by glucose-6-phosphate, for which the yeast enzyme is insensitive (Sols 1976). The relevance of the reported in vitro inhibition of yeast hexokinase by trehalose-6-phosphate for the control of glucose influx into glycolysis in vivo remains unclear. In the present paper we show that ggsl/tpsl mutants are extremely sensitive to low glucose levels even in the presence of a 100-fold higher level of galactose. We also show that the GGS1/TPS1 gene is required continuously during exponential growth on glucose in spite of the absence of trehalose under such conditions.
Materials and methods
Construction of ggsl/tpsl A and tps2A strains. The coding region of the GGS1/TPS1 gene was deleted completely in vitro using a PCR approach and replaced by the TRP1 gene. For this purpose the
112
GGS1/TPSI gene was subcloned into Ylplac211 on a 3.6-kb HindIII/SphI fragment. Two primers hybridizing to sequences at the start and the end of the coding region and inserting a BgIII site were used to amplify the whole plasmid without the coding region. The PCR product was digested with BglII and ligated. The resulting plasmid was again linearized with BgIII and the TRP1 gene was inserted on a 0.85-kb BamHI fragment derived from plasmid YDpW (Betben et al. 1991). The deletion of the genomic copy was accomplished by one-step gene disruption (Rothstein 1983) after digesting the plasmid with KpnI and PvuII. The mutant allelesfdpI and bypl-3 were cloned by plasmid eviction, cifl was isolated using PCR (Van Aelst et al. 1993). The glc6 allele was kindly provided by John Cannon (Columbia, Mich.). All the alleles were subcloned on a 4.8-kb HindIII/BamHI fragment into Ylplac211 (Gietz and Sugino 1988). The resulting plasmids were linearised with SphI and integrated into the yeast genome downstream from ggsl/tpsIA: :TRP1. Singlecopy integration was confirmed by Southern analysis. The integration of the plasmid created a tandem arrangement of the duplicated GGS1/TPS1 upstream sequence interrupted by the TRP1 gene and the integrated plasmid containing the URA3 gene. Spontaneous loss of the URA3 marker was selected for by using a medium containing 5-Fluoro-orotic acid (FOA) (Sherman et al, 1986). Ura- clones were checked for concomitant loss of the TRP1 gene which indicated that the two copies of the upstream sequences had recombined to cut out all the intervening sequences and to move the integrated GGS1/TPS1 mutant allele in place of the deletion allele. This was confirmed by Southern analysis and by the sequencing of PCR products derived from genomic DNA to show the presence of the respective mutations. The strains obtained in this way and the other strains used in the present study are shown in Table 1. The TPS2 gene was deleted using a similar approach. TPS2 (De Virgilio et al. 1993) was subcloned on a 3.9-kb SacI/ClaI fragment into pUC19 (digested with SacI and AccI) and the coding region was deleted and replaced with a BglII site by PCR as described above. The LEU2 marker derived from YDpL (Berben et al. 1991) was inserted into the BglII site on a BamHI fragment. The resulting plasmid was digested with SacI and HindIII prior to yeast transformation.
Determination of trehalose, trehalose-6-phosphate synthase, fructose-l,6-bisphosphatase and glycogen synthase. Trehalose was determined essentially as described by Neves et al. (1991). Trehalose6-phosphate synthase activity was determined according to De Virgilio et al. (1990), fructose-l,6-bisphosphatase according to Gancedo and Gancedo (1971 ), and glycogen synthase according to Frangois et al. (1988). Protein was determined with the microbiuret method (Zamenhoff 1957).
Plasmid stability test. The GGSI/TPS1 gene was subcloned on a 3.6kb HindIII/SphI fragment (Van Aelst et al. 1993) into vector YRp7 (Struhl et al. 1979) giving plasmid YRp7GGS1/TPS1. YRp7 is an autonomously replicating plasmid lacking, however, both centromeric or 2 pm sequences and thus is very unstable even under selective conditions (Zakian and Kupfer 1982; Murray and Szostak 1983). It carries TRP1 as selective marker for propagation in yeast. The W3031A strain carrying the ggsl/tpslA: : LEU2 mutation (Van Aelst et al. 1993) was transformed with this plasmid and transformants were selected on synthetic medium (Sherman et al. 1986) lacking tryptophan and containing galactose as a carbon source. Complementation of the ggsl/tpslA mutation for growth on fermentable sugars was confirmed by replica plating onto glucose medium. No complementation of the growth defect was observed with the vector alone. Individual transformants carrying the vector or the plasmid YRp7GGS1/TPS1 were grown in liquid synthetic medium lacking tryptophan and containing galactose as carbon source (SDtrpGal) into early stationary phase (approximately 5x107 cells/ml). These conditions are selective for the plasmid, but not for the insert. Ten microliters of this culture were transferred into 5 ml of fresh medium. The media used were again SDtrpGal (selection for the vector), YP with 2% galactose (YPGal; non-selective) and YP with 8% glucose (YPD 8%; test conditions, plasmid with GGS1/TPS1 only). The cultures were allowed to grow for about ten generations. The number of cells in the culture was determined both by direct counting under the microscope and by plating appropriate dilutions onto YPGal. Both methods gave consistent results showing that the cells remained viable even without the plasmid. The proportion of plasmid-containing cells was determined by replica-plating the colonies from the YPGal plates onto either SDtrpGaI or YPD. For transformants with the plasmid YRp7GGS1/TPS1 all the colonies that were tryptophan prototrophic were also glucose-positive and vice versa showing that no suppressor mutations or revertants had accumulated. Ten microliters of the cultures grown in YPD 8 % were transferred into the same fresh medium and grown again for about ten generations and the proportion of plasmid-carrying cells was determined as above (indicated as '2.growth' in Table 3). As a control that the plasmid had not integrated into the genome but could still be lost rapidly, 10 pl of the YPD 8%-grown cultures were inoculated into YPGal medium and grown two times for about ten generations and the number of cells carrying the plasmid was determined. A number of individual colonies still containing the plasmid was then further checked for plasmid instability.
Results Sporulation of homozygous ggs l/tps l A and tps2 A diploids. S. cerevisiae strains were diploidized by transformation with a centromeric vector (YCp50, marker URA3) carrying the HO gene (Herskowitz and Jensen 1991). Single-cell clones of transformants were checked for their ploidy by flow cytometry using propidium iodide as a DNA stain (Popolo et al. I982). Diploids were then streaked out on FOA medium (Sherman et al. 1986) to select for plasmid loss which was confirmed by replica plating onto uracil-free medium. The strains obtained in this way are shown in Table 1. Diploid strains were pregrown in 5 ml of YP medium (Sherman et al. 1986) with 2% galacrose until either logarithmic or stationary phase. Cells were sedimented, resuspended in 5 ml of KAc medium (1% KAc, pH 6.0) and shaken for 4 days at 24 ~ The number of cells that entered meiosis was counted relative to those that did not.
Glucose sensitivity and uptake measurements. The cells to be used for glucose sensitivity measurements were grown up overnight in YP galactose or YP ethanol medium and subsequently inoculated in 100 ml of synthetic medium containing 2% galactose or 3% ethanol at 0D6o o of about 0.1. After 2 h of incubation at 30~ glucose was added in the indicated concentrations. Growth was monitored by measuring the OD6oo every 20 min for 3 h. Glucose uptake was measured in Mes (4-Morpholine ethanesulfonic acid)/KOH buffer (pH 6.0) for periods of 10 s as described by Ramos et al. (1988).
P r o p e r t i e s o f the d i f f e r e n t p o i n t m u t a n t s in the s a m e genetic background W e h a v e c l o n e d t h e f d p l , bypl, glc6 and cifl a l l e l e s o f the GGS1/TPS1 g e n e and e x c h a n g e d t h e m for the o r i g i n a l w i l d - t y p e a l l e l e in t h r e e d i f f e r e n t w i l d - t y p e strains: YSH6.36-3B, a haploid descendant of the M5 diploid strain), W 3 0 3 - 1 A , and SP1. T h e p h e n o t y p e o f the r e s u l t ing strains w a s c o m p a r e d w i t h that o f the g g s l / t p s l A m u tant. E x c e p t for the glc6 allele, all m u t a n t a l l e l e s c a u s e d a g r o w t h d e f e c t on m e d i a c o n t a i n i n g g l u c o s e or f r u c t o s e (Fig. 1). T h e e f f e c t w a s m o s t p r o n o u n c e d in W 3 0 3 - 1 A and SP1. In the M 5 b a c k g r o u n d the g r o w t h d e f i c i e n c y w a s less s t r i n g e n t for all alleles. T h e b y p l m u t a n t started g r o w i n g on g l u c o s e and f r u c t o s e after a lag, as r e p o r t e d p r e v i o u s l y ( H o h m a n n et al. 1992), w h i l e the cifl m u t a n t g r e w to s o m e e x t e n t on g l u c o s e b u t n o t on f r u c t o s e , as r e p o r t e d p r e v i o u s l y for the o r i g i n a l i s o l a t e ( N a v o n et al. 1979; C h a r l a b et al. 1985). E v e n g g s l / t p s l A and f d p l s h o w e d r e s i d u a l
113 l
b
a T
1 S. cerevisiae strains used in this study
Strain
Genotype
Source and/or reference
W303-1A
MATa teu2-3/112 ura3-1 trpl-1 his3-11~15 ade2-1 canl-lO0 GAL SUC2
Thomas and Rothstein 1989
Strains isogenic to W303-1A: YSH290 ggsl/tpslA: : TRP1 YSH272 ggsl/tpslA: : LEU2 YSH322 fdpl YSH321 bypl-3 YSH430 cij7 YSH431 gIc6 YSH377 ggsl/tpslA: : TRPt hxtlA: : URA3 YSH379 ggsl/tpslA: : TRP1 hxtlA: : URA3 snf3A: :HIS3 YSH352 ggsl/tpslA: : TRP1 ragl/hxt4A: : URA3 YSH354 ggsl/tpslA: : TRP1 snf3A: :HIS3 ragl/hxt4A: : URA3 YSH312 ggsl/tpslA: : TRP1 hxk2A: : LEU2 YSH450 tps2A: : LEU2 YSH310 hxk2A: : LEU2 YSH327 hxklA: : HIS3 hxk2A: : LEU2 YSH339 MATc~ YSH370 MATo~ ggslA: : LEU2 snf3A: : HIS3 Diploid strains isogenic to W303-1A: MA Ta/MA T a YSH337 ggsl/tpslA//ggsl/tpslA (YSH290 diploidized) YSH340 tps2A/tps2A (YSH450 diploidized) YSH555 fdpl/fdpl (YSH322 diploidized) YSH552 bypl-3/bypI-3 (YSH321 diploidized) YSH551 cifl/cifl (YSH430 diploidized) YSH553 glc6/glc6 (YSH431 diptoidized) YSH554 ggsl/tpslz~ hxk2A//ggsl/tpslA hxk2A (YSH312 diploidized) YSH341 MATa leu2 his3 trpl ade8 canl ura3 SP1
M. Wigler (Toda et al. 1985)
Strains isogenic to SPI: YSH289 ggsl/tpslA: : TRP1 YSH324 fdpI YSH323 bypI-3 YSH432 cifl YSH433 glc6 Hohmann et al. 1993 YSH6.36.-3B ATCC90756 MATa leu2-3/112 ura3-52 trpl-92 SUC GAL (Tiffs strain is a haploid derivative of the diploid strain M5: Schaaff et al. t989) Strains isogenic to YSH6.36.-3B: YSH292 ggslA: : TRP1 YSH326 fdpl YSH325 bypl-3 YSH428 cifl YSH429 gIc6 YS H455 tps2A: : LEU2 Diploid strains: MATla/MATcx leu2-3/112 ura3-52 trpl-92 his4 GAL SUC/Ieu2-3/ll2 ura3-52 trpl-92 GAL M5 (YSH143) Diploid strains isogenic to YSH6.36,-3B: YSH557 ggsl/tpslA/ggsl/tpslA (YSH292 diploidized) YSH559 fdpl/fdpl (YSH326 diploidized) YSH558 bypl/bypI (YSH325 diploidized) YSIt560 cifl/c~fl (YSH428 diploidized) YSH561 glc6/glc6 (YSH429 diploidized) YSH562 tps2A/tps2A (YSH455 diploidized)
growth on glucose in the M5 background, w e have reported previously that antimycin A suppresses the partial growth deficiency on glucose of the b y p l mutant (Hohmann et al. 1992). We now show that antimycin A has the same suppressive effect on the growth deficiency caused by the other alleles, both on a glucose- and fructose-containing medium. However, the suppressive effect is also dependent
on the genetic background (Fig. 1). It is most pronounced in M5, less in the W303-1A background and is absent in SP1. In diploid strains, the growth deficiency caused by the g g s l / t p s l A mutation is much weaker. A homozygous g g s l / t p s l A diploid strain grew without an appreciable lag phase on glucose, but did not grow on fructose medium (data not shown). A homozygous g g s l / t p s l A h x k 2 A dip-
114
131
-g E
6
+ o
03
03
N
0.8
Fig. 1 Growth of the different ggsl/tpsl mutants as influenced by the genetic background9 Genetic backgrounds are from left to right: M5 (YSH6.36.-3B), W303-1A and SPI. Relevant genotype (allele of GGS1/TPS1) from top to bottom: wild-type, ggslA/tpslA, fdpl, bypl-3, cifl, and glc6. Cells were pregrown on YP galactose, replica plated onto YP medium with 2% glucose or fructose, containing 2 mg/1 of antimycin A where indicated, and grown overnight at 30 ~
0.6
~.~ 0.4 o
loid strain grew on glucose medium like the corresponding haploid strain (Hohmann et al. 1993) but, as opposed to the haploid strain, it also grew to some extent on fructose medium (data not shown). Trehalose-6-phosphate synthase activity was strongly reduced in the three genetic backgrounds by all ggsl/tpsl alleles, except for the glc6 allele which had no effect at all (Fig. 2, upper panel). Residual activity was highest in the W303-1A strain. A similar situation was observed for the trehalose level (Fig. 2, lower panel) except that the bypl mutant in the M5 background had a significantly higher trehalose level than the other alleles, as reported previously . gback-i (Hohmann et al. 1992, 1994). In the other genetic grounds, however, there was no difference with the other alleles. The glc6 mutant had a significantly higher trehalose level in the W303-1A and SP1 backgrounds. Inactivation of fructose-l,6-bisphosphatase (FbPase) bY addition of glucose to cells growing on a non-fermentable carbon source was studied in the isogenic series of ggsl/tpsl mutants in the W303-1A (Fig. 3) and M5 (data not shown) backgrounds. With all mutant alleles, except again for the glc6 allele, FbPase inactivation was absent.
0.2
0.0
~
~
~
~
e
~
~
o~
N
F 2 Trehalose-6-phosphate synthase specific activity (upper panel) and trehalose content (lower panel) of ggsl/tpsl mutants in different genetic backgrounds (M5, W303-1A or SP1, as indicated)
Sporulation defect in
ggsl/tpsl mutants
We have reported previously that several diploid ggsl/tpsl mutants show a severe sporulation deficiency (Van Aelst et al. 1993). We now show that this is true for all alleles in two different backgrounds, W303-1A and M5, except
115 I
I
I
I
I
l
I
40
Table 2 Percentage of sporulation in diploid homozygous ggsl/tps] strains. Transfer of cells in logarithmic or stationary (data not shown) phase to sporulation medium gave the same results, with the exception of the tps2 diploid strain
ol
E E
g
30
20
25
10
o
, 0
20
40
60
80
100
120
time (rain)
g n
i
F 3 Glucose-induced inactivation of fructose-1,6-bisphosphatase i cells growing on a non-fermentable carbon source. Strains: 9
Genotype
% Sporulation
W303-lA wild-type a/~ W303- l A ggsl/tpslA//ggsl/tpsl A W303- l A tps2A/tps2A
30
