catabolite modulator factor: a possible mediator of catabolite repression in bacteria

Catabolite Modulator Factor: A Possible Mediator of Catabolite Repression in BacteriaAuthor(s): Agnes Ullmann, Francoise Tillier and Jacques MonodSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 73, No. 10 (Oct., 1976), pp. 3476-3479Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/66622 .

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Proc. Natl. Acad. Sci. USA Vol. 73, No. 10, pp. 3476-3479, October 1976 Biochemistry

Catabolite modulator factor: A possible mediator of catabolite repression in bacteria

(physiological repression and derepression/f5-galactosidase/adenosine 3':5'-cyclic monophosphate)

AGNES ULLMANN, FRANCOISE TILLIER, AND JACQUES MONOD*

Service de Biochimie Cellulaire, Institut Pasteur, 75015 Paris, France

Contributed by Jacques Monod, July 21, 1976

ABSTRACT Water soluble extracts of Escherichia coli cells have been found to exert an extremely strong repressive effect upon the expression of catabolite sensitive operons. The compound responsible for this activity has been partially purified and proves to be of low molecular weight and heat stable. The effect of this compound, hereafter designated as catabolite modulator factor, is only partially antagonized by adenosine 3':5'-cyclic monophosphate. The possible role of catabolite modulator factor in the physiological regulation of catabolite repression is discussed.

The inhibitory effect of glucose or other carbohydrates on the expression of certain operons is a well known phenomenon called catabolite repression (1). It is currently believed today that adenosine 3':5'-cyclic monophosphate (cAMP) and its receptor protein are the sole physiological mediators of this effect. During the last few years different lines of evidence suggested that cAMP might not be the unique regulator of catabolite repression (2). In our search for mediators, other than cAMP, we found that water soluble extracts of Escherichia coi cells exerted strong repression upon catabolite sensitive operons. In the present paper, we describe some of the physiological properties of the so far only partially purified compound which appears to be responsible for this effect, and discuss its possible role in the regulation of catabolite repression.

MATERIALS AND METHODS

Strains and Media. E. coi K-12 wild-type strain 3000 was currently used in these studies. Strain CA8224.1 (L8UV5, lac promoter mutant) and cya mutant 283 were generously given to us by Jon Beckwith. Strain L8UV5 cya was constructed by transducing strain cya 283 to lace with PI-phage lysate grown on L8UV5. Minimal media 63 (KH2PO4 0.1 MS NH4Cl 20 mM, MgSO4 1 mM, FeCl3 1 AM, pH 7) and 68 (Tris-HCl 0.1 M, KH2PO4 1 mM, NH4Cl 20 mM, MgSO4 1 mM, FeCls 1 AM, pH 7.5) supplemented with vitamin B1 and different carbon sources have been used throughout this study.

Extraction and Partial Purification of Catabolite Modu- lator Factor. A culture of strain 3000 grown overnight in medium 68 with glucose (0.4%) as a carbon source, was centrifuged at low speed. The bacterial pellet was resuspended in distilled water previously adjusted to pH 8 (25 mg of dry weight bacteria per ml of H20). The suspension was kept in a boiling-water bath for 12 min. After cooling, the suspension was immediately centrifuged at 20,000 X g for 10 min. The supernatant repre- sents crude catabolite modulator factor (CMF). A purification of about 10-50 times (based on the loss of UV absorbing material) has been achieved through the following steps: (i) passage

Abbreviations: cAMP, adenosine 3':5'-cyclic monophosphate; CMF, catabolite modulator factor; IPTG, isopropyl-o-D-thiogalactoside. * Deceased.

on a Dowex AG 1X8 (Bio-Rad) column. Crude CMF (100 ml) is diluted with two volumes of water and deposited on a Dowex column which is in the acetate form (100 ml of resin). The active material is not retained on the column and is washed off by H20 until disappearance of UV absorbing material in the eluant is noted.

(ii) The eluted material is concentrated almost to dryness under vacuum. To the residue (about 10 ml) 200 ml of ethanol are added and the mixture is kept overnight at 00. The pre- cipitate which is formed is eliminated by centrifugation; the supernatant is evaporated under vacuum and the residue re- dissolved in water (Dowex-ethanol fraction). Ten microliters of the Dowex-ethanol fraction corresponds to the amount of CMF obtained from approximately 1 mg (dry weight) of bacteria.

Physiological Catabolite Repression and Derepression. Experiments meant to test effectors of catabolite repression are usually conducted by comparing results observed during growth in the presence of carbohydrates differing in their repression effects such as glucose (which gives fairly strong repression) and glycerol (which exhibits only weak repression). For our purposes, we needed to explore the full range of the catabolite repression effect. Now it has been repeatedly observed (cf. ref. 1) that in absence of added specific effectors the level of catabolite repression is entirely modulated by the relative availability of a carbon source versus a source of nitrogen. By severely limiting the nitrogen source while the carbon source is in excess, extreme repression (95% or more) is observed as compared with the reverse condition where the availability of the carbon source is limited and the nitrogen source is in excess. Under the latter condition, the differential rates of enzyme synthesis observed are significantly higher than is ever observed in the presence of cAMP; therefore we will call it "full physiological derepression."

Conditions of Extreme Physiological Repression and Derepression. E. coli K-12 cannot use urea as nitrogen source nor sucrose as carbon source. If urease is added concomitantly with urea, NH3 will be liberated, thus allowing bacterial growth. To obtain growth in the presence of sucrose (as sole carbon source), we have to add invertase to obtain glucose lib- eration.

To obtain maximal repression, we add a rapidly metabolized carbon source (glucose) at saturating concentrations and urea as the sole nitrogen source. In the presence of sufficiently low concentrations of urease, the growth of bacteria will be limited by the availability of the liberated NH3.

For maximal derepression, (NH4)2SO4 is used as nitrogen source and sucrose as the carbon source. Correctly adjusting the invertase concentrations produces a severe growth limitation.

Under both conditions, for obvious reasons, growth is linear

3476



Biochemistry: Ullmann et al. Proc. Natl. Acad. Sci. USA 73(1976) 3477

OD

0.7 1/

1000.

o ~~~~~~~0.3 - L

0

50 60 min 0

o \ 0

10 20 30 40 pl CMF

FIG. 1. Effect of CMF on ,B-galactosidase synthesis. To exponentially growing cultures of strain 3000 in medium 68-B1 with glucose (0.4%) as carbon source, we added different amounts of CMF (Dowex ethanol fraction) simultaneously with 1 mM IPTG. After two generations, growth was stopped, and the amount of ,B-galactosidase determined in toluenized suspensions. The insert of the figure shows growth curves in the absence (X-X) and in the presence of 10 (0 -*), 20 (A-,A), and 40 (0-0) ,al/ml of CMF. GZ refers to ,B-galactosidase.

and the linear slope is proportional to the concentration of enzyme added.

It will be immediately seen that under such conditions the growth-rate, as usually defined (i.e., under conditions of exponential growth) decreases constantly as the bacterial mass increases. For purposes of comparison between different experimental conditions, therefore, it is essential that the initial bacterial densities as well as mass increases should be identi- cal.

The experiments were actually carried out as follows: 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-Bi-glucose medium (for repression studies) or in glucose-free 63-B1 medium (for studying derepression). After complete exhaustion of the re- sidual (NH4)2SO4 or glucose the cultures were diluted in order to obtain 108 bacteria per ml. For nitrogen-limitation experiments, 0.1% urea and different concentrations of urease are added; carbon-source linmtation is carried out in the presence of 0.4% sucrose + invertase. As soon as linear growth obtained the cultures were induced with 1 mM isopropyl-f-D-thiogalactoside (IPTG).

Enzymatic Assays. -f-Galactosidase (f-D-galactoside ga- lactohydrolase, EC 3.2.1.23) was assayed according to Pardee, Jacob, and Monod (3) and tryptophanase [L-tryptophan in- dole-lyase, (deaminating), EC 4.1.99.1] was measured as described by Newton et al. (4).

Reagents and Enzymes. IPTG, cAMP, urease, and invertase were purchased from Sigma Chemical Co., L-tryptophan from Calbiochem, and all other chemicals from Merck.

RESULTS

General properties of catabolite modulator factor (CMF)

When partially purified extract, containing CMF, is added to an exponentially growing culture of wild-type strain 3000 the synthesis of f-galactosidase is strongly affected. As it can be seen in Fig. 1, the effect depends on the amount of CMF added. At the higher concentrations of CMF, extreme repression is observed (amounting to 99% of the levels attained by a culture

Table 1. Effect of CMF on the differential rate of ,B-galactosidase and tryptophanase synthesis

CMF 3-Galactosidase Tryptophanase (,Vl/ml) (units/mg) (%) (units/mg) (%)

3900 100 250 100 50 1060 27 113 45

100 540 14 22 9 200 155 4 8.1 3

To exponentially growing cultures of strain 3000 in medium 68-B, with glycerol (0.4%) as the carbon source, we added different a-mounts of crude CMF as well as IPTG (mM) and tryptophan (2 mg/ml). After two generations, growth was stopped and the amount of enzymes were determined in toluenized suspensions. The results are expressed as units of enzyme per mg of bacteria dry weight.

under conditions of full physiological derepression). Thus, the range of repression which can be obtained by addition of CMF appears to be as wide as can be observed also under our so-called conditions of extreme "physiological repression."

CMF has little effect on bacterial growth (see insert of Fig. 1). This is in itself an indication that CMF does not significantly affect the expression of any of the operons involved in the synthesis of biosynthetic enzymes.

Table 1 shows the comparative effect of CMF on the synthesis of f-galactosidase and tryptophanase. It can be seen that the synthesis of both enzymes is strongly inhibited. Further studies on several other systems known to be catabolite inde- pendent (A. Dessein, Thesis; A. Dessein, F. Tillier, and A. Ull- mann, manuscript in preparation) have shown that the synthesis of glucose-6-phosphate dehydrogenase and phosphogluco- mutase are strictly not affected by the CMF, while the rate of synthesis of amylomaltase and galactokinase (known to be catabolite sensitive) are strongly repressed. Therefore it appears that the CMF acts specifically on the expression of catabolite- sensitive operons.

When the action of CMF is studied over a longer period of tiime, the repression effect is seen to disappear and the initial differential rate of enzyme synthesis is restored (Fig. 2). At this tiime, CMF cannot be detected anymore in bacterial superna- tants (data not shown) suggesting that it has been metabolized.

We have no indications as to the chemical nature of CMF; however, the following points concerning its general properties may be recorded: (i) it is a small molecular weight product (molecular weight less than 1000) since it is retained on a Se- phadex G-10 column. (ii) It has no apparent charge (it is not retained on any anionic or cationic exchange columns) at var- ioius pHs and ionic strengths. (iii) It is heat, alkali, and acid stable (it is stable at 1000 in the presence of 1 M HCI or 1 M NaOH).

Catabolite modulator factor and cAMP

While the antagonistic effect of cAMP towards catabolite repression is well established, a disturbing feature of this phenomenon is that, under conditions of extreme repression (nitrogen source limitation for instance), cAMP only partially reverts catabolite repression (5). Because in the presence of CM4F, catabolite sensitive enzymes can be repressed as severely as under conditions of nitrogen source limitation, it was of interest to determine to what extent cAMP can overcome such effects. Table 2 shows an experiment where the level of catabolite repression was modulated either by "physiological repression" or by addition of CMF. At high levels of repression w]hether obtained under physiological repression or by addition



3478 Biochemistry: Ullmann et al. Proc. Natl. Acad. Sci. USA 73(1976)

1.5 -

rO / o 0 j _ 1.0 -/

0.5

0/

250 500 ; g/ml

FIG. 2. Kinetics of CMF inhibition of 3-galactosidase synthesis. An exponentially growing culture of strain 3000 in medium 68-B1 with glucose (0.4%) as the carbon source was divided; one part received IPTG (1 mM) (0--); the other, IPTG + CMF (0-0). At different time intervals thereafter, the amount of 3-galactosidase was determined. 3-Galactosidase activity (U) is represented as a function of bacterial cell mass.

of CMF the rates of enzyme synthesis observed in the presence of cAMP are 70-90% lower than the rates which obtain under conditions of full physiological derepression. The fact that cAMP behaves in the same way under both conditions strongly suggests that repression obtained by CMF is similar to "physiological repression."

Catabolite modulator factor and lactose promoter mutant

Strain L8UV5 is a lactose promoter mutant (6) which, under the conditions used to test it, was found insensitive to catabolite repression and to the effect of cAMP (7). It was therefore of particular interest to test whether this mutant would respond at all to extreme conditions of physiological repression or de-

Table 2. Effect of cAMP on "physiological repression" and on repression exerted by CMF

Rate of bacterial mass in- GZ GZ

Urease crease cAMP (units/ CMF cAMP (units/ (jg/ml) (ag/hr) added mg) (Ml/ml) added mg)

- -* - 2650 - + 2400 - -* + 9000 - 6300

0.4 43 - 1430 50 + 1190 0.4 39 + 7800 50 - 4500 0.26 30 - 590 100 + 510 0.26 26 + 6000 100 - 3200 0.13 19 - 265 200 + 138 0.13 19 + 3200 200 - 1600

Experimental conditions for "physiological repression" are described in Materials and Methods. Conditions for repression obtained by CMF are as described for Table 1. Final concentration of cAMP was 5 mM. GZ refers to n-galactosidase. Full physiological derepression was 16,000 units of GZ per mg of bacteria. * Exponential growth.

Table 3. Effect of cAMP and "physiological derepression" on strains 3000 and L8UV5

Strain Addition Units/mg of GZ

3000 Glucose 4,600 Glucose + cAMP 9,000 Sucrose + invertase 15,800

L8UV5 Glucose 4,670 Glucose + cAMP 5,300 Sucrose + invertase 11,400

Experimental conditions for "physiological derepression" are described in Materials and Methods. The concentration of invertase was 0.1 Ag/ml and the rate of mass increase for both strains was 10 pg/hr. cAMP (5 mM) was added to exponentially growing cultures in medium 63-Bi. IPTG was used at a final concentration of 1 mM. GZ refers to ,B-galactosidase.

repression, and show sensitivity to CMF. Table 3 shows that mutant L8UV5, while responding only slightly to cAMP, can be further largely derepressed under our physiological conditions. Fig. 3 shows that L8UV5 mutant can be strongly repressed by addition of CMF, which shows, however, much less sensi- tiyity than the wild-type organism. Moreover, at high concentrations of CMF, it appears to be completely insensitive to the antagonistic effect of cAMP. Furthermore, we have tested the properties of this mutant when the lactose promoter gene was transduced into an adenylcyclase negative (cya) strain, where it shows the same sensitivity to CMF. These results would appear strongly to suggest that cAMP could hardly be a unique modulator of catabolite repression in vivo.

DISCUSSION

Since the discovery that cAMP is an antagonist of permanent (8) and transient (9) catabolite repression, extensive studies carried out on in vitro systems (10-13) resulted in a coherent model of positive regulation exerted by cAMP and its receptor protein.

On this basis, it has generally been concluded that modulations in the intracellular concentration of cAMP could entirely account for catabolite repression effects as observed under

o Ay s \~AT

2.5

, I I1 .

50 I00 200 LI CMF

FIG. 3. Effect of CMF on the differential rate of 3-galactosidase synthesis in strain L8UV5. Experimental conditions were the same as described in the legend of Fig. 1. L8UV5 cya+ (o-0) in the absence and (Q-A ) in the presence of cyclicA MP; LgUVs cya- (- .) in the absence and (v-v) in the presence of cAMP. cAMP concentration was 5 mM.



Biochemistry: Ullmann et al. Proc. Natl. Acad. Sci. USA 73 (1976) 3479

various conditions in vivo. Some conflicting observations prompted us to search for other or further types of mediators or modulators of these effects. The physiological (in vivo) properties of CMF as described in the present paper, would seem to indicate it as a good candidate for such a mediator.

As we have seen, partially purified extracts of CMF: (i) specifically repress the expression of catabolite sensitive operons without showing any effects on catabolite insensitive systems; (ii) show effects similar in range to those that obtain under extreme conditions of physiological repression; (iii) have an apparent metabolism of CMF by the cells, which would allow rapid modulations of its intracellular concentration. Further- more, (iv) a promoter mutant, largely insensitive to cAMP, proves to be sensitive both to physiological derepression and to the repressive effect of CMF.

At this point, our data do not allow proposing an actual model for the mechanism of action of CMF. We should perhaps point out the fact that our results do not identify the active compound present in water soluble extract as being directly responsible for the effects observed. These effects might be due to another compound or compounds derived from CMF through its metabolism in vivo. While preliminary, the experimental evidence does very strongly suggest that besides the cyclic AMP-cAMP receptor protein-promoter interaction, another mechanism does operate in the cell to control the activity of catabolite sensitive systems.

We wish to thank Dr. Maxime Schwartz for many helpful discussions and Dr. Jon Beckwith for the generous gift of strains. This research was

supported by grants from the Delegation Generale a la Recherche Scientifique et Technique, the Centre National de la Recherche Scientifique, and the National Institutes of Health.

1. Magasanik, B. (1961) cold Spring Harbor Symp. Quant. Biol. 26, 249-256.

2. Ullmann, A. (1974) Biochem. Biophys. Res. Commun. 57, 348-352.

3. Pardee, A. B., Jacob, F. & Monod, J. (1959) J. Mol. Biol. 1, 165-178.

4. Newton, W. A., Morino, Y. & Snell, E. E. (1965) J. Biol. Chem. 240, 1211-1218.

5. Ullmann, A., Contesse, G., Crepin, M., Gros, F. & Monod, J. (1969) in Fogarty International Center Proceedings, eds. Kall, T. W., Rotbell, M. & Condliffe, P. (National Institutes of Health, Bethesda, Md.), Vol. 4, pp. 215-246.

6. Silverstone, A. E., Arditti, R. & Magasanik, B. (1970) Proc. Natl. Acad. Sci. USA 66, 773-779.

7. Beckwith, J., Grodzicker, T. & Arditti, R. (1972) J. Mol. Biol. .69, 155-160.

3. Ullmann, A. & Monod, J. (1968) FEBS Lett. 2,57-60. !3. Perlman, R. & Pastan, I. (1968) Biochem. Biophys. Res. Commun.

30, 656-664. 10. Emmer, M., De Crombrugghe, B., Pastan, I. & Perlman, R. (1970)

Proc. Natl. Acad. Sci. USA 66, 480-487. 11. Zubay, G., Schwartz, D. & Beckwith, J. (1970) Proc. Natl. Acad.

Sci. USA 66, 104-110. 12. Wu, F. Y.-H., Nath, K. & Wu, C. W. (1974) Biochemistry 12,

2567-2572. 13. Wu, C. W. & Wu, F. Y.-H. (1974) Biochemistry 12, 2573-

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catabolite modulator factor: a possible mediator of catabolite repression in bacteria

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