Molec. gen. Genet. i62, 83-87 (1978) © by Springer-Verlag 1978
Catabolite Repression in Escherichia coli Mutants Lacking Cyclic AMP* Alain Dessein, Maxime Schwartz, and Agn~s U l l m a n n D6partements de Biologic Mol6culaire et de Biochimie et G6n6tique Microbienne, Institut Pasteur, F-75724 Paris Cedex 15 (France)
Summary. The regulation of catab, olite repression o f /~-galactosidase has been studied in Escherichia coli mutants deleted for the adenyl cyclase gene (CyaA) , and thus unable to synthesize cyclic A M P . It has been f o u n d that, provided a second m u t a t i o n occurs either in the crp gene coding for the catabolite gene activator protein (CAP) or in the Lactose region, these mutants exhibit catabolite repression. If the catabolite repression seen in the m u t a n t strains corresponds to the mechanism operating in wild-type cells, the results would suggest that the intracellular concentration o f cyclic A M P c a n n o t be the unique regulator o f catabolite repression.
Introduction M a n y enzymes responsible for the catabolism of carb o n c o m p o u n d s in a variety of microorganisms are repressed by glucose or its degradation products. This p h e n o m e n o n described in 1947 by M o n o d ( M o n o d , 1947) for the effect o f glucose on the f o r m a t i o n o f /~-galactosidase was generalized and called catabolite repression by Magasanik (Magasanik, 1961). C a t a b o lite repression occurs when a c o m p o u n d is m e t a b o lized so rapidly that the level of catabolites is, greater than required for biosynthetic reactions. M a n y aspects of the molecular basis of this repression have been elucidated. N o w it is currently believed that catabolite repression in bacteria is mediated by cyclic A M P and its intracellular receptor ( C A P protein). The cyclic A M P - C A P protein-pro* Jacques Monod was still with us when most of the work described in this and the following paper was accomplished. His constant interest, his unfailing advice, his warm support, were invaluable. It will he difficult for us to ever enjoy a successful experiment without regretting that he cannot share this pleasure with us. For offprints contact." A. Dessein
moter interaction allows for an efficient transcription ofcatabolite sensitive operons. According to this model the extent of catabolite repression is uniquely dependent u p o n the intracellular concentration of cyclic AMP. One of the strongest arguments in favor of this model is the fact that in adenyl cyclase deficient mutants (cya) the expression o f catabolite sensitive operons is dramatically reduced and can be restored by addition of cyclic A M P . We have searched for second site mutations enabling cya mutants to grow on one or several carbohydrates in the absence of cyclic AMP. In the present paper, we describe the behavior of these double mutants with respect to catabolite repression. We show that under different physiological conditions, they can be catabolically repressed or derepressed (the term o f c a t a b o l i t e repression, being used according to the definition given by Magasanik (Magasanik, 1961)).
Materials and Methods Strains and Media
The E. coli strains used during this work are listed in Table 1. Minimal medium 63 (KHgPO4, 0.1 M; NH,CI, 20mM; MgSO4, 1 mM; FeCL3, 1 ~M; pH 7) was supplemented with vitamin B1 (1 ~tg/ml), different carbon sources (0.4%) and in some experiments with cAMP (5 raM). Solid medium contained Difco Agar (15%) and different carbon sources at final concentration of 0.4%. Reagents and Enzymes
Isopropyl fi-D-thiogalactoside (IPTG), adenosine Y-5"cyclic monophosphate (cAMP) urease and invertase were purchased from Sigma Chemical Company. Bactotryptone, Yeast extract and Agar Difco from Difco Lab., N-methyl-N'-nitro-N-nitrosoguanidine from Eastman and all other chemicals from Merck.
0026-8925/78/0162/0083/$01.00
A. Dessein et al. : Catabolite Repression in E. coli M u t a n t s Lacking Cyclic A M P
84
Table 1 Strains
Genotype b and relevant characters
Source
3000 3000 YA597 Hfr G61 AB2151 CA8404 CA8445 Ca8449 TIT 1 TIT 2 TIT 3 TIT 101
thi thi, ilv, lac aroB, h i s thi, ilv, laeZ4 leu, strA thi, cya d, crpo ~ thi, cyaa, crp45 thi, cyaA, crp49 thi, ilv thi, ilv, mal T thi, ilv, aroB thi, cya A
TIT TIT TIT TIT TIT TIT TIT TIT TIT TIT
thi, thi, thi, thi, thi, thi, thi, thi, thi, thi,
Dept. Biol. M o l 6 c u l a i r e - I n s t i t u t Pasteur Dept. Biol. M o l ~ c u l a i r e - I n s t i t u t Pasteur Dept. Biol. M o l 6 c u l a i r e - I n s t i t u t Pasteur Dept. Biol. M o l 6 c u l a i r e - I n s t i t u t Pasteur (D. Sabourin, J. Beckwith, 1975) (D. Sabourin, J. Beckwith, 1975) (D. Sabourin, J. Beckwith, 1975) F r o m 300 YA597 by transduction with Ply (3000) 2 R M a l - spontaneous m u t a n t s o f TIT 1 F r o m TIT 2 by transduction with Ply (Hfr G61) F r o m 3000 transduced to A r a - 2 R by P1~ (CA8404) (as described by Brickman et al., 1975) F r o m TIT 3 transduced to Ileu + M a l - by Ply (CA8404) F r o m AB2151 transduced to Ileu + M a l - by Ply (CA8404) This paper This paper This paper This paper This paper This paper This paper This paper
102 103 201 202 203" 302 303 308 310 311
cyaz, aroB cya~, lacZ H, leu, strA cya~, lacAD1 cyaA, laCAD2 cyaA, Lac + cyaa, crPAD2 cya~, crpAD3 cya A, crpAD8 cya~, crpADW cflaA, crpADl I
a The mutation yielding the Lac + phenotype was not mapped in this strain b All strains are Hfr (but Hfr character of strain 3000 YA597 was not tested)
Enzymatic Assays /Lgalactosidase (/LD-galactoside galacto-hydrolase E.C. 32.1.23) was assayed according to Pardee, Jacob and M o n o d (1959) in toluenized cell suspensions. The differential rate of/~-galactosidase synthesis is expressed as units of enzyme per m g of dry weight bacteria. Extreme physiological repression and derepression were obtained using conditions as described by U l l m a n n et al. (1976).
Isolation o f the Mutants E. coli strain TIT 101, carrying a deletion of the cya gene, is unable to grow using as sole carbon source lactose, maltose, glycerol or arabinose, because the synthesis of the enzymes implicated in the first steps of the degradation of these carbohydrates are under control of catabolite repression. F r o m this strain, we have isolated two types of m u t a n t s : m u t a n t s exhibiting a Lac + phenotype; m u t a n t s exhibiting a Lac +, Mal +, Ara + and Glp + phenotype.
Isolation o f the cya 3 Lac + Mutants TIT 101 cells growing exponentially in L-medium were washed once with 63B1 medium and plated on 63 B~ Agar m e d i u m containing lactose as sole carbon source. After 48 h Lac + colonies appeared at a frequency of 10 7. These m u t a n t s are unable to grow on maltose, glycerol or arabinose as carbon source.
Isolation o f cya~ Mal +, Lac + Mutants Without mutagenesis we did not obtain any cya A Mal + Lac + mutants.
Strain TIT 101 was mutagenized with N-methyl-N'-nitro-Nnitrosoguanidine as described by Miller (1972a) and plated immediately on 63B1 medium containing maltose as sole carbon source. Mal + colonies appeared with a frequency of 10 -6. Fifty m u t a n t s have been tested for their ability to grow on other carbohydrates. All of them metabolize lactose, glycerol and arabinose in the absence of cAMP.
Mapping All transductions were carried out using bacteriophage Ply as described by Miller (1972b).
1. Cya d Lac + Strains. At least 100 Lac + transductants obtained from the transduction of strain TIT 103 (cya~, LacZ~) by P1 phage grown on each of the cya A Lac +, strains TIT 201 and TIT 202 were selected on lactose-cAMP medium. On lactose minimal medium, all these transductants are Lac + even in the absence of cAMP. Therefore it appears that the mutations conferring the Lac + phenotype to TIT 201 and TIT 202 m u t a n t s are closely linked to Lac genes. They are likely to be promoter m u t a n t s as are the Lac + revertants of strain crp- isolated by Arditti et al. (1973). 2. CyaA Mal + Strains. Pleiotropic carbohydrate pseudo revertants of a strain carrying a deletion of the cya gene have already been isolated by L. Soll: strain CA 8404 (in Sabourin and Beckwith (1975)). The mutations have been localized in the crp gene and called crp*. Two approaches have been used to localize the mutations conferring the Mal +, Lac +, Ara +, Glp + phenotype to strains TIT302, 303, 310 and 311. a) Since crp is 30% cotransducible with aroB lysates of phage P1 grown on the above strains were used to transduce strain TIT 102 (cyaa, aroB): 20% to 30% of AroB + transductants were Mal + ,
A. Dessein et al. : Catabolite Repression in E. coli M u t a n t s Lacking Cyclic A M P
Results
Table 2. Differential rate of /~-galactosidase synthesis in Lac + pseudo revertants Strains
Glucose
Glucose+cAMP
Lactose
3000 TIT 101 TIT 201 TIT 202 TIT 203
2,500 200 1,600 1,000 500
6,000 10,000 13,000 11,000 9,000
3,500 a 1,700 1,200 500
85
Study of Lac + Revertants of cya d Strain Spontaneous Lac + pseudo revertants were isolated from strain T I T 101, which carries a deletion in gene cya. The corresponding mutations are tightly linked to the lac operon (see Materials and Methods) and are most probably located in the lac protomer. When the cells are grown in minimal glucose medium the induced level of/%galactosidase was about 2-8 times higher in the Lac ÷ revertants than in the cya~ parent, and can still be increased more than 10 fold by adding cAMP. (Table 2). Extreme repression and derepression conditions can be obtained if the nitrogen or carbon sources are limited. This was achieved by using the ureaurease or sucrose-invertase technique (Ullmann et al., 1976). The behaviour of the cya~ strain and its Lac + pseudo revertants under these extreme conditions is shown in Table 3. It can be seen that by limiting the nitrogen source a significant repression is obtained while carbon source limitation has practically no effect on the rate of enzyme synthesis. It is noteworthy that the parental cyae strain cannot be derepressed in either of these
Does not grow on this carbon source. The results are expressed in units of/?-galactosidase per m g dry weight bacteria. Strains were grown in 63 B1 minimal medium supplemented with 0.4% glucose or 0.4% lactose a n d induced with 1 m M I P T G for two generations of growth. Final concentration of c A M P was 5 m M
Ara ÷, G l p - , Lac ÷. If transduction is carried out with a lysate of phage P1 grown on CA 8404, the percentage of AroB +, Mal +, Ara + transductants is about the same. b) The same P1 lysates were used to transduce the Mal + character to a strain deleted in crp and cya genes (CA 8445 and CA 8449). At least one h u n d r e d transductants selected on maltose-cAMP minimal m e d i u m are Mal ÷, Ara ÷, Glp ÷ Lac + even in the absence of cAMP.. Therefore it appears that the mutations conferring the Mal ÷, Ara ÷, Glp +, Lac + phenotype to TIT 302, 303, 308, 310 and 311 mutants are located in gene crp or very close to it.
Table 3. Differential rate of/~-galactosidase synthesis in Lac ÷ pseudo revertants under conditions of extreme repression and derepression Conditions o f extreme repression Urease (gg/ml)
_ 0.14 0.07
Rate of bacterial mass increase (gg/h)
a 25 l0
Strain 3000
Strain TIT 101 -cAMP
1.2 x 10 - 4 c A M P
4,000 800 300
160 b b
3,900 2,050 810
Strain 3000
Strain TIT 101
Strain TIT 201
Strain TIT 202
1,600 1,260 700
1,100 760 450
Strain TIT 201
Strain TIT 202
1,600 1,700 2,000
1,100 1,200 1,300
Conditions of extreme Derepression Invertase (gg/ml)
-
0.125 0.075
Rate of bacterial mass increase (gg/h)
-" 50 25
3,600 16,500 18,000
-cAMP
9 x 10- 5 c A M P
160 185 190
1,800 3,800 5,000
a Exponential growth. b Not done. The results are expressed in units of fl-galactosidase per mg dry weight bacteria. Overnight cultures grown in 63 medium supplemented with vitamin B1 and 0.4% glucose were centrifuged, washed and resuspended either in (NH4)2SO4 free 63-Bl-glucose medium (for repression studies) or in glucose-free 63-Bl-medium (for derepression studies). After complete exhaustion of the residual (NH4)2SO4 or glucose, the cultures were diluted in order to obtain l0 s bacterial, per ml. For nitrogen limitation experiments: 0.1% urea and different concentrations of urease were added; carbon-source limitation is carried out in the presence of 0.4% sucrose+invertase. As soon as linear growth was obtained the cultures were induced with 1 m M isopropyl-fi-D-thiogalactoside (IPTG). Growth was stopped when the cultures attained 2 x 108 bacteria per ml.
A. Dessein et al. : Catabolite Repression in E. coli Mutants Lacking Cyclic A M P
86
the most significant finding is that in glycerol or succinate containing media the enzyme synthesis is derepressed to the same extent as in the presence of cyclic AMP (Table 4). Furthermore a 3 fold derepression of/~-galactosidase is observed in the mutants when extreme conditions of derepression are used (Table 5). Surprisingly however no repression is observed if the nitrogen source is limited.
Table 4. Differential rate of/~-galactosidase synthesis in crp* mutants Strains
Glucose
G l u c o s e + c A M P Glycerol
Succinate
3000 TIT 101 TIT 302 TIT 308 TIT 310 TIT 311
2,500 200 5,800 6,300 7,200 6,000
6,000 10,000 9,700 10,500 9,500 9,000
11,000 _a 10,000 12,500 14,000 15,500
6,500 a 10,200 10,500 11,500 11,700
Discussion
a Does not grow on this carbon source, The results are expressed in units of/~-galactosidase per mg dry weight bacteria. Strains were grown in 63 B1 minimal medium supplemented with 0.4% glucose, 0.4% glycerol or 0.1% succinate and induced with I m M IPTG for two generations of growth
We have shown that two types of pseudo revertants isolated from a cya A strain still exhibit catabolite repression to a certain extent. Lac + revertants, presumably carrying a mutation in the lac promoter, can still be repressed using conditions of nitrogen starvation known to ensure extreme catabolite repression in the wild type strain. Pleiotropic carbohydrate positive revertants, presumably carrying a mutation in crp, can still be derepressed under conditions of carbon source limitation known to ensure extreme derepression. Provided that the catabolite repression seen in the mutant strain corresponds to the mechanism operating in the wild type cells our results show that a regulation of catabolite repression can take place in E. coli strains lacking cyclic AMP. No detectable amount of cyclic AMP could be found in the cya~ strain (Epstein, in Brickman et al., 1973), and the same is likely to be true in the two types of pseudo revertants. Therefore this nucleotide can not be responsible for the modulation of catabolite repression observed in these strains, and it is tempting to speculate that at least another effector
conditions unless cyclic AMP is added at a concentration allowing a 5-10 fold increase in the rate of enzyme synthesis.
Study o f M a l + Ara ÷ L a c + Revertants o f a cya~ Strain
Pleiotropic Mal + Ara ÷ Lac + revertants could only be obtained after mutagenesis (see Materials and Methods) and at a low frequency. They probably therefore carry a very specific mutation or perhaps a double mutation. Transduction experiments demonstrate that the mutation(s) is (are) in gene crp, or very tightly linked to it. In minimal glucose medium the revertants synthesize about 30 times more/~-galactosidase than the cya 4 parent strain, and 2-3 times more than the wild type cya + strain (Table 4). But Table 5. Differential rate of/?-galactosidase synthesis in crp* mutants under conditions of extreme repression or derepression
Conditions of extreme repression Urease (gg/ml)
Rate of bacterial mass increase (gg/h)
0.14 0.07
- ~ 25 10
Strains 3000
4,000 800 300
Strain TIT 302
6,000 5,000 4,600
Strain TIT 310
7,500 6,600 7,600
Conditions of extreme derepression
a Exponential growth. The results are expressed in units of ~-galactosidase per mg dry weight bacteria. Experimental conditions as described in the legend of Table 3
Invertase Rate of bacterial (ixg/ml) mass increase (gg/h)
Strain 3000
Strain TIT 302
Strain TIT 310
_ 0.15 0.125 0.075
3,600 10,000 16,500 18,000
4,000 8,000 11,000 14,000
7,500 7,500 10,000 21,000
a 60 50 25
A. Dessein et al. : Catabolite Repression in E. coli Mutants Lacking Cyclic AMP is i n v o l v e d in the process. A g o o d c a n d i d a t e for such an effector c o u l d be the c a t a b o l i t e m o d u l a t o r factor ( C M F ) ( U l l m a n n et al., 1976). It w o u l d seem t h a t d e r e p r e s s i o n requires the presence o f " a c t i v a t e d " C A P protein, w h e t h e r the activation results f r o m the presence o f c A M P , or f r o m a crp* m u t a n t . I n d e e d a c y a A strain is n o t d e r e p r e s s e d u n d e r c o n d i t i o n s o f c a r b o n source l i m i t a t i o n unless s u b o p t i m a l a m o u n t o f c A M P is p r e s e n t in the med i u m ( T a b l e 3) b u t crp* r e v e r t a n t s are d e r e p r e s s i b l e even in the a b s e n c e o f c A M P . L a c ÷ r e v e r t a n t s (prom o t e r m u t a n t s ) c a n n o t be d e r e p r e s s e d : they exhibit wild t y p e C A P p r o t e i n a n d no c A M P is synthesized in these m u t a n t s . These findings w o u l d fit a simple m o d e l involving a d o u b l e r e g u l a t i o n o f C A P p r o t e i n which w o u l d be a c t i v a t e d by c A M P a n d i n h i b i t e d by C M F . Such a model, however, w o u l d n o t readilly a c c o u n t for the fact t h a t crp* m u t a n t s are n o t repressible. In c o n c l u s i o n we believe that o u r results, showing t h a t c a t a b o l i t e r e p r e s s i o n can be m o d u l a t e d in strains c a r r y i n g a d e l e t i o n o f a d e n y l cyclase gene, strongly suggest that the v a r i a t i o n o f i n t r a c e l l u l a r c o n c e n t r a tion o f cyclic A M P c a n n o t be c o n s i d e r e d as the u n i q u e r e g u l a t o r y m e c h a n i s m for the c o n t r o l o f c a t a b olite sensitive systems. It seems likely t h a t at least one o t h e r effector is involved, p e r h a p s C M F , p o s s i b l y acting on the C A P protein. Acknowledgments. We wish to thank Jon Beckwith for the generous
gift of strains.
87
This work has been supported by grants from the °' D616gation Gbn6rale ~ la Recherche Scientifique et Technique" and the "Centre National de la Recherche Scientifique (Laboratoire Associ~ n ° 270)".
References Arditti, R., Grozzicker, T., Beckwith, J.: Cyclic adenosine monophosphate-independent mutants of the lactose operon of Eseherichia coll. J. Bact. 114, 652 655 (1973) Brickman, E., Soll, L., Beckwith, J.: Genetic characterization of mutations which affect catabolite-sensitive operon in Eseherichia coli, including deletions of the gene for adenylcyclase. J. Bact. 116, 582-587 (1973) Magasanik, B.: Catabolite repression. Cold Spr. Hath. Syrup. quant. Biol. 26, 249 (1961) Miller, J.H.: Experiments in molecular genetics, pp. 44~48. New York: Cold Spring Harbor Lab. 1972a Miller, J.H. : Experiments in molecular genetics, pp. 125. New York : Cold Spring Harbor Lab. 1972b Monod, J.: The phenomenon of enzymatic adaptation. Growth 11,223 (1947) Pardee, A.B., Jacob, F., Monod, J. : The genetic control and cytoplasmic expression of"inductibility" in the synthesis of/~-galactosidase by Escherichia coli. J. molec. Biol. 1, 165-178 (1959) Sabourin, D., Beckwith, J. : Deletion of Escherichia coli crp genes. J. Bact. 122, 338-340 (1975) Ullmann, A., Tillier, F., Monod, J. : Catabolite modulator factor. A possible mediator of catabolite repression in bacteria. Proc. nat. Acad. Sci. (Wash.) 73, 3476-3479 (1976) C o m m u n i c a t e d by F. G r o s Received September 26, 1977
Catabolite Repression in Escherichia coli Mutants Lacking Cyclic AMP* Alain Dessein, Maxime Schwartz, and Agn~s U l l m a n n D6partements de Biologic Mol6culaire et de Biochimie et G6n6tique Microbienne, Institut Pasteur, F-75724 Paris Cedex 15 (France)
Summary. The regulation of catab, olite repression o f /~-galactosidase has been studied in Escherichia coli mutants deleted for the adenyl cyclase gene (CyaA) , and thus unable to synthesize cyclic A M P . It has been f o u n d that, provided a second m u t a t i o n occurs either in the crp gene coding for the catabolite gene activator protein (CAP) or in the Lactose region, these mutants exhibit catabolite repression. If the catabolite repression seen in the m u t a n t strains corresponds to the mechanism operating in wild-type cells, the results would suggest that the intracellular concentration o f cyclic A M P c a n n o t be the unique regulator o f catabolite repression.
Introduction M a n y enzymes responsible for the catabolism of carb o n c o m p o u n d s in a variety of microorganisms are repressed by glucose or its degradation products. This p h e n o m e n o n described in 1947 by M o n o d ( M o n o d , 1947) for the effect o f glucose on the f o r m a t i o n o f /~-galactosidase was generalized and called catabolite repression by Magasanik (Magasanik, 1961). C a t a b o lite repression occurs when a c o m p o u n d is m e t a b o lized so rapidly that the level of catabolites is, greater than required for biosynthetic reactions. M a n y aspects of the molecular basis of this repression have been elucidated. N o w it is currently believed that catabolite repression in bacteria is mediated by cyclic A M P and its intracellular receptor ( C A P protein). The cyclic A M P - C A P protein-pro* Jacques Monod was still with us when most of the work described in this and the following paper was accomplished. His constant interest, his unfailing advice, his warm support, were invaluable. It will he difficult for us to ever enjoy a successful experiment without regretting that he cannot share this pleasure with us. For offprints contact." A. Dessein
moter interaction allows for an efficient transcription ofcatabolite sensitive operons. According to this model the extent of catabolite repression is uniquely dependent u p o n the intracellular concentration of cyclic AMP. One of the strongest arguments in favor of this model is the fact that in adenyl cyclase deficient mutants (cya) the expression o f catabolite sensitive operons is dramatically reduced and can be restored by addition of cyclic A M P . We have searched for second site mutations enabling cya mutants to grow on one or several carbohydrates in the absence of cyclic AMP. In the present paper, we describe the behavior of these double mutants with respect to catabolite repression. We show that under different physiological conditions, they can be catabolically repressed or derepressed (the term o f c a t a b o l i t e repression, being used according to the definition given by Magasanik (Magasanik, 1961)).
Materials and Methods Strains and Media
The E. coli strains used during this work are listed in Table 1. Minimal medium 63 (KHgPO4, 0.1 M; NH,CI, 20mM; MgSO4, 1 mM; FeCL3, 1 ~M; pH 7) was supplemented with vitamin B1 (1 ~tg/ml), different carbon sources (0.4%) and in some experiments with cAMP (5 raM). Solid medium contained Difco Agar (15%) and different carbon sources at final concentration of 0.4%. Reagents and Enzymes
Isopropyl fi-D-thiogalactoside (IPTG), adenosine Y-5"cyclic monophosphate (cAMP) urease and invertase were purchased from Sigma Chemical Company. Bactotryptone, Yeast extract and Agar Difco from Difco Lab., N-methyl-N'-nitro-N-nitrosoguanidine from Eastman and all other chemicals from Merck.
0026-8925/78/0162/0083/$01.00
A. Dessein et al. : Catabolite Repression in E. coli M u t a n t s Lacking Cyclic A M P
84
Table 1 Strains
Genotype b and relevant characters
Source
3000 3000 YA597 Hfr G61 AB2151 CA8404 CA8445 Ca8449 TIT 1 TIT 2 TIT 3 TIT 101
thi thi, ilv, lac aroB, h i s thi, ilv, laeZ4 leu, strA thi, cya d, crpo ~ thi, cyaa, crp45 thi, cyaA, crp49 thi, ilv thi, ilv, mal T thi, ilv, aroB thi, cya A
TIT TIT TIT TIT TIT TIT TIT TIT TIT TIT
thi, thi, thi, thi, thi, thi, thi, thi, thi, thi,
Dept. Biol. M o l 6 c u l a i r e - I n s t i t u t Pasteur Dept. Biol. M o l ~ c u l a i r e - I n s t i t u t Pasteur Dept. Biol. M o l 6 c u l a i r e - I n s t i t u t Pasteur Dept. Biol. M o l 6 c u l a i r e - I n s t i t u t Pasteur (D. Sabourin, J. Beckwith, 1975) (D. Sabourin, J. Beckwith, 1975) (D. Sabourin, J. Beckwith, 1975) F r o m 300 YA597 by transduction with Ply (3000) 2 R M a l - spontaneous m u t a n t s o f TIT 1 F r o m TIT 2 by transduction with Ply (Hfr G61) F r o m 3000 transduced to A r a - 2 R by P1~ (CA8404) (as described by Brickman et al., 1975) F r o m TIT 3 transduced to Ileu + M a l - by Ply (CA8404) F r o m AB2151 transduced to Ileu + M a l - by Ply (CA8404) This paper This paper This paper This paper This paper This paper This paper This paper
102 103 201 202 203" 302 303 308 310 311
cyaz, aroB cya~, lacZ H, leu, strA cya~, lacAD1 cyaA, laCAD2 cyaA, Lac + cyaa, crPAD2 cya~, crpAD3 cya A, crpAD8 cya~, crpADW cflaA, crpADl I
a The mutation yielding the Lac + phenotype was not mapped in this strain b All strains are Hfr (but Hfr character of strain 3000 YA597 was not tested)
Enzymatic Assays /Lgalactosidase (/LD-galactoside galacto-hydrolase E.C. 32.1.23) was assayed according to Pardee, Jacob and M o n o d (1959) in toluenized cell suspensions. The differential rate of/~-galactosidase synthesis is expressed as units of enzyme per m g of dry weight bacteria. Extreme physiological repression and derepression were obtained using conditions as described by U l l m a n n et al. (1976).
Isolation o f the Mutants E. coli strain TIT 101, carrying a deletion of the cya gene, is unable to grow using as sole carbon source lactose, maltose, glycerol or arabinose, because the synthesis of the enzymes implicated in the first steps of the degradation of these carbohydrates are under control of catabolite repression. F r o m this strain, we have isolated two types of m u t a n t s : m u t a n t s exhibiting a Lac + phenotype; m u t a n t s exhibiting a Lac +, Mal +, Ara + and Glp + phenotype.
Isolation o f the cya 3 Lac + Mutants TIT 101 cells growing exponentially in L-medium were washed once with 63B1 medium and plated on 63 B~ Agar m e d i u m containing lactose as sole carbon source. After 48 h Lac + colonies appeared at a frequency of 10 7. These m u t a n t s are unable to grow on maltose, glycerol or arabinose as carbon source.
Isolation o f cya~ Mal +, Lac + Mutants Without mutagenesis we did not obtain any cya A Mal + Lac + mutants.
Strain TIT 101 was mutagenized with N-methyl-N'-nitro-Nnitrosoguanidine as described by Miller (1972a) and plated immediately on 63B1 medium containing maltose as sole carbon source. Mal + colonies appeared with a frequency of 10 -6. Fifty m u t a n t s have been tested for their ability to grow on other carbohydrates. All of them metabolize lactose, glycerol and arabinose in the absence of cAMP.
Mapping All transductions were carried out using bacteriophage Ply as described by Miller (1972b).
1. Cya d Lac + Strains. At least 100 Lac + transductants obtained from the transduction of strain TIT 103 (cya~, LacZ~) by P1 phage grown on each of the cya A Lac +, strains TIT 201 and TIT 202 were selected on lactose-cAMP medium. On lactose minimal medium, all these transductants are Lac + even in the absence of cAMP. Therefore it appears that the mutations conferring the Lac + phenotype to TIT 201 and TIT 202 m u t a n t s are closely linked to Lac genes. They are likely to be promoter m u t a n t s as are the Lac + revertants of strain crp- isolated by Arditti et al. (1973). 2. CyaA Mal + Strains. Pleiotropic carbohydrate pseudo revertants of a strain carrying a deletion of the cya gene have already been isolated by L. Soll: strain CA 8404 (in Sabourin and Beckwith (1975)). The mutations have been localized in the crp gene and called crp*. Two approaches have been used to localize the mutations conferring the Mal +, Lac +, Ara +, Glp + phenotype to strains TIT302, 303, 310 and 311. a) Since crp is 30% cotransducible with aroB lysates of phage P1 grown on the above strains were used to transduce strain TIT 102 (cyaa, aroB): 20% to 30% of AroB + transductants were Mal + ,
A. Dessein et al. : Catabolite Repression in E. coli M u t a n t s Lacking Cyclic A M P
Results
Table 2. Differential rate of /~-galactosidase synthesis in Lac + pseudo revertants Strains
Glucose
Glucose+cAMP
Lactose
3000 TIT 101 TIT 201 TIT 202 TIT 203
2,500 200 1,600 1,000 500
6,000 10,000 13,000 11,000 9,000
3,500 a 1,700 1,200 500
85
Study of Lac + Revertants of cya d Strain Spontaneous Lac + pseudo revertants were isolated from strain T I T 101, which carries a deletion in gene cya. The corresponding mutations are tightly linked to the lac operon (see Materials and Methods) and are most probably located in the lac protomer. When the cells are grown in minimal glucose medium the induced level of/%galactosidase was about 2-8 times higher in the Lac ÷ revertants than in the cya~ parent, and can still be increased more than 10 fold by adding cAMP. (Table 2). Extreme repression and derepression conditions can be obtained if the nitrogen or carbon sources are limited. This was achieved by using the ureaurease or sucrose-invertase technique (Ullmann et al., 1976). The behaviour of the cya~ strain and its Lac + pseudo revertants under these extreme conditions is shown in Table 3. It can be seen that by limiting the nitrogen source a significant repression is obtained while carbon source limitation has practically no effect on the rate of enzyme synthesis. It is noteworthy that the parental cyae strain cannot be derepressed in either of these
Does not grow on this carbon source. The results are expressed in units of/?-galactosidase per m g dry weight bacteria. Strains were grown in 63 B1 minimal medium supplemented with 0.4% glucose or 0.4% lactose a n d induced with 1 m M I P T G for two generations of growth. Final concentration of c A M P was 5 m M
Ara ÷, G l p - , Lac ÷. If transduction is carried out with a lysate of phage P1 grown on CA 8404, the percentage of AroB +, Mal +, Ara + transductants is about the same. b) The same P1 lysates were used to transduce the Mal + character to a strain deleted in crp and cya genes (CA 8445 and CA 8449). At least one h u n d r e d transductants selected on maltose-cAMP minimal m e d i u m are Mal ÷, Ara ÷, Glp ÷ Lac + even in the absence of cAMP.. Therefore it appears that the mutations conferring the Mal ÷, Ara ÷, Glp +, Lac + phenotype to TIT 302, 303, 308, 310 and 311 mutants are located in gene crp or very close to it.
Table 3. Differential rate of/~-galactosidase synthesis in Lac ÷ pseudo revertants under conditions of extreme repression and derepression Conditions o f extreme repression Urease (gg/ml)
_ 0.14 0.07
Rate of bacterial mass increase (gg/h)
a 25 l0
Strain 3000
Strain TIT 101 -cAMP
1.2 x 10 - 4 c A M P
4,000 800 300
160 b b
3,900 2,050 810
Strain 3000
Strain TIT 101
Strain TIT 201
Strain TIT 202
1,600 1,260 700
1,100 760 450
Strain TIT 201
Strain TIT 202
1,600 1,700 2,000
1,100 1,200 1,300
Conditions of extreme Derepression Invertase (gg/ml)
-
0.125 0.075
Rate of bacterial mass increase (gg/h)
-" 50 25
3,600 16,500 18,000
-cAMP
9 x 10- 5 c A M P
160 185 190
1,800 3,800 5,000
a Exponential growth. b Not done. The results are expressed in units of fl-galactosidase per mg dry weight bacteria. Overnight cultures grown in 63 medium supplemented with vitamin B1 and 0.4% glucose were centrifuged, washed and resuspended either in (NH4)2SO4 free 63-Bl-glucose medium (for repression studies) or in glucose-free 63-Bl-medium (for derepression studies). After complete exhaustion of the residual (NH4)2SO4 or glucose, the cultures were diluted in order to obtain l0 s bacterial, per ml. For nitrogen limitation experiments: 0.1% urea and different concentrations of urease were added; carbon-source limitation is carried out in the presence of 0.4% sucrose+invertase. As soon as linear growth was obtained the cultures were induced with 1 m M isopropyl-fi-D-thiogalactoside (IPTG). Growth was stopped when the cultures attained 2 x 108 bacteria per ml.
A. Dessein et al. : Catabolite Repression in E. coli Mutants Lacking Cyclic A M P
86
the most significant finding is that in glycerol or succinate containing media the enzyme synthesis is derepressed to the same extent as in the presence of cyclic AMP (Table 4). Furthermore a 3 fold derepression of/~-galactosidase is observed in the mutants when extreme conditions of derepression are used (Table 5). Surprisingly however no repression is observed if the nitrogen source is limited.
Table 4. Differential rate of/~-galactosidase synthesis in crp* mutants Strains
Glucose
G l u c o s e + c A M P Glycerol
Succinate
3000 TIT 101 TIT 302 TIT 308 TIT 310 TIT 311
2,500 200 5,800 6,300 7,200 6,000
6,000 10,000 9,700 10,500 9,500 9,000
11,000 _a 10,000 12,500 14,000 15,500
6,500 a 10,200 10,500 11,500 11,700
Discussion
a Does not grow on this carbon source, The results are expressed in units of/~-galactosidase per mg dry weight bacteria. Strains were grown in 63 B1 minimal medium supplemented with 0.4% glucose, 0.4% glycerol or 0.1% succinate and induced with I m M IPTG for two generations of growth
We have shown that two types of pseudo revertants isolated from a cya A strain still exhibit catabolite repression to a certain extent. Lac + revertants, presumably carrying a mutation in the lac promoter, can still be repressed using conditions of nitrogen starvation known to ensure extreme catabolite repression in the wild type strain. Pleiotropic carbohydrate positive revertants, presumably carrying a mutation in crp, can still be derepressed under conditions of carbon source limitation known to ensure extreme derepression. Provided that the catabolite repression seen in the mutant strain corresponds to the mechanism operating in the wild type cells our results show that a regulation of catabolite repression can take place in E. coli strains lacking cyclic AMP. No detectable amount of cyclic AMP could be found in the cya~ strain (Epstein, in Brickman et al., 1973), and the same is likely to be true in the two types of pseudo revertants. Therefore this nucleotide can not be responsible for the modulation of catabolite repression observed in these strains, and it is tempting to speculate that at least another effector
conditions unless cyclic AMP is added at a concentration allowing a 5-10 fold increase in the rate of enzyme synthesis.
Study o f M a l + Ara ÷ L a c + Revertants o f a cya~ Strain
Pleiotropic Mal + Ara ÷ Lac + revertants could only be obtained after mutagenesis (see Materials and Methods) and at a low frequency. They probably therefore carry a very specific mutation or perhaps a double mutation. Transduction experiments demonstrate that the mutation(s) is (are) in gene crp, or very tightly linked to it. In minimal glucose medium the revertants synthesize about 30 times more/~-galactosidase than the cya 4 parent strain, and 2-3 times more than the wild type cya + strain (Table 4). But Table 5. Differential rate of/?-galactosidase synthesis in crp* mutants under conditions of extreme repression or derepression
Conditions of extreme repression Urease (gg/ml)
Rate of bacterial mass increase (gg/h)
0.14 0.07
- ~ 25 10
Strains 3000
4,000 800 300
Strain TIT 302
6,000 5,000 4,600
Strain TIT 310
7,500 6,600 7,600
Conditions of extreme derepression
a Exponential growth. The results are expressed in units of ~-galactosidase per mg dry weight bacteria. Experimental conditions as described in the legend of Table 3
Invertase Rate of bacterial (ixg/ml) mass increase (gg/h)
Strain 3000
Strain TIT 302
Strain TIT 310
_ 0.15 0.125 0.075
3,600 10,000 16,500 18,000
4,000 8,000 11,000 14,000
7,500 7,500 10,000 21,000
a 60 50 25
A. Dessein et al. : Catabolite Repression in E. coli Mutants Lacking Cyclic AMP is i n v o l v e d in the process. A g o o d c a n d i d a t e for such an effector c o u l d be the c a t a b o l i t e m o d u l a t o r factor ( C M F ) ( U l l m a n n et al., 1976). It w o u l d seem t h a t d e r e p r e s s i o n requires the presence o f " a c t i v a t e d " C A P protein, w h e t h e r the activation results f r o m the presence o f c A M P , or f r o m a crp* m u t a n t . I n d e e d a c y a A strain is n o t d e r e p r e s s e d u n d e r c o n d i t i o n s o f c a r b o n source l i m i t a t i o n unless s u b o p t i m a l a m o u n t o f c A M P is p r e s e n t in the med i u m ( T a b l e 3) b u t crp* r e v e r t a n t s are d e r e p r e s s i b l e even in the a b s e n c e o f c A M P . L a c ÷ r e v e r t a n t s (prom o t e r m u t a n t s ) c a n n o t be d e r e p r e s s e d : they exhibit wild t y p e C A P p r o t e i n a n d no c A M P is synthesized in these m u t a n t s . These findings w o u l d fit a simple m o d e l involving a d o u b l e r e g u l a t i o n o f C A P p r o t e i n which w o u l d be a c t i v a t e d by c A M P a n d i n h i b i t e d by C M F . Such a model, however, w o u l d n o t readilly a c c o u n t for the fact t h a t crp* m u t a n t s are n o t repressible. In c o n c l u s i o n we believe that o u r results, showing t h a t c a t a b o l i t e r e p r e s s i o n can be m o d u l a t e d in strains c a r r y i n g a d e l e t i o n o f a d e n y l cyclase gene, strongly suggest that the v a r i a t i o n o f i n t r a c e l l u l a r c o n c e n t r a tion o f cyclic A M P c a n n o t be c o n s i d e r e d as the u n i q u e r e g u l a t o r y m e c h a n i s m for the c o n t r o l o f c a t a b olite sensitive systems. It seems likely t h a t at least one o t h e r effector is involved, p e r h a p s C M F , p o s s i b l y acting on the C A P protein. Acknowledgments. We wish to thank Jon Beckwith for the generous
gift of strains.
87
This work has been supported by grants from the °' D616gation Gbn6rale ~ la Recherche Scientifique et Technique" and the "Centre National de la Recherche Scientifique (Laboratoire Associ~ n ° 270)".
References Arditti, R., Grozzicker, T., Beckwith, J.: Cyclic adenosine monophosphate-independent mutants of the lactose operon of Eseherichia coll. J. Bact. 114, 652 655 (1973) Brickman, E., Soll, L., Beckwith, J.: Genetic characterization of mutations which affect catabolite-sensitive operon in Eseherichia coli, including deletions of the gene for adenylcyclase. J. Bact. 116, 582-587 (1973) Magasanik, B.: Catabolite repression. Cold Spr. Hath. Syrup. quant. Biol. 26, 249 (1961) Miller, J.H.: Experiments in molecular genetics, pp. 44~48. New York: Cold Spring Harbor Lab. 1972a Miller, J.H. : Experiments in molecular genetics, pp. 125. New York : Cold Spring Harbor Lab. 1972b Monod, J.: The phenomenon of enzymatic adaptation. Growth 11,223 (1947) Pardee, A.B., Jacob, F., Monod, J. : The genetic control and cytoplasmic expression of"inductibility" in the synthesis of/~-galactosidase by Escherichia coli. J. molec. Biol. 1, 165-178 (1959) Sabourin, D., Beckwith, J. : Deletion of Escherichia coli crp genes. J. Bact. 122, 338-340 (1975) Ullmann, A., Tillier, F., Monod, J. : Catabolite modulator factor. A possible mediator of catabolite repression in bacteria. Proc. nat. Acad. Sci. (Wash.) 73, 3476-3479 (1976) C o m m u n i c a t e d by F. G r o s Received September 26, 1977
