Unstable inducer-specific repressor for β-galactosidase in Escherichia coli

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UNSTABLE INDUCER-SPECIFIC REPRESSOR FOR tJ-GALACTOSIDASE IN ESCHERICHIA COLI

D. JOE CLARK' Un,iversity of Copenhagen, Copenhagen (Denmark) (Received January 26th, I965)

SUMMARY The rate of formation and decay of inducer-specific repressor for tJ-galactosidase (p-n-galactoside galactohydrolase, EC 3.2.1.23) has been examined using sub-saturating concentrations of inducer to "titrate" for repressor. The rate of formation of inducer-specific repressor for p-galactosidase was assayed by the change in the differential rate of ,B-galactosidase synthesis during a shift from succinate-restricted growth to succinate-unrestricted growth. The new steady-state repressed rate of unrestricted growth is attained within 0.6 doublings, The rate of decay of inducer-specific repressor for .B-galactosidase was examined by the change in the differential rate of ,B-galactosidase synthesis during a shift from succinate-unrestricted growth to succinate-restricted growth. The new steady-state derepressed rate of restricted growth is attained within 0.3 doublings, indicating that inducer-specific repressor is unstable. A comparison with the constitutive strain indicates that derepression during such a shift is unique to the inducible state and thus is probably due to the disappearance of inducer-specific repressor.

INTRODUCTION Existence of a repressor which prevents the expression of a cistron or operon is implied by dominance ofthe unexpressed state in inducible systems'. Althoughearly experiments by PARnEE2 suggested that the repressor for p-galactosidase (p-D-galactoside galactohydrolase, EC 3.2. I.23) is not a protein more recent evidence indicates that repressor is a proteins:". NovIcK8 and CLARK9 have described methods for the examination of the repressor of p-galactosidase synthesis. The method of NOVICK depends upon the appearance or disappearance of repressor during shifts from one temperature to another in mutants unable to form repressor at high temperatures. The method of CLARK employs sub-saturating concentrations of inducer which inactivate fractions of the Abbreviation: IPTG, isopropyl-tJ-D-thiolgalactopyranoside. , Fellow of National Institutes of Health, U.S.A. (I963-1965)· Present addres: Virus Laboratory, University of California, Berkeley, Calif. (U.S.A.).

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total repressor. Shifts in growth conditions which are known to lead to different steady-state levels of repression are used to examine the appearance or disappearance of repressor. The latter method considers effects due to catabolite repression and subdivides the repression system into: (I) a part which is antagonized by inducer called "inducer-specific repression"; and (a) a part which is not antagonized by inducer called "inducer-independent repression". NOVICK analyzed the stability of repressor by examination of the rate of increase in the differential rate of ,B-galactosidase formation in a mutant which is phenotypically inducible at 30° and which is phenotypically constitutive at 43°. The shift from 30-43° is accompanied by a gradual increase in the differential rate of ,B-galactosidase formation which he interpreted as being due to the decay of existing repressor. The lag in enzyme formation was used to indicate the degree of stability of the existing product of the i-gene. The lag was much greater than one generation indicating repressor decay is dependent on growth. Evidence is presented in this paper which shows that decay of inducer-specific repressor is not growth-dependent and repressor is unstable.

MATERIALS AND METHODS

Bacterial strains The cryptic (y-) strains of Escherichia coli used in these experiments include: ML 3 which is phenotypically lacr, inducible for ,B-galactosidase and. presumably i+o+z+y-; and ML 35 which is phenotypically lacr , constitutive for ,a-galactosidase and presumably i-o+z+y-. ML strains were obtained from Dr. J. MONOD. Culture conditions Cells were grown in basal-salts medium (composition in gil: KH 2P0 4, 13.6; NH 4Cl, 1.1; MgS0 4'7 H 20, 0.25; FeCl a, 0.0016; the pH was adjusted to 7.0) to which the appropriate carbon source and nutrients were added prior to inoculation. Experiments were initiated from cultures which had grown at least four generations at 30° after growth overnight in carbon-limited cultures. Succinate-restricted growth was obtained by the slow but constant addition of succinate to a carbon-limited culture as previously described", Measurement of growth Growth was measured by an increase in the absorbance at 450 mfJ- in r-em cuvettes with a Zeiss Model PQM II spectrophotometer. Unit absorbance corresponds to approx . zoo fJ-g dry wt. of cells per ml, The yield of ML 3 is approx. 1.0 absorbance unit per 440 f1.g of succinate per ml. Protein synthesis was measured by L-[14C]histidine incorporation. One ml of the cell suspension was added to 3 ml of cold 7.5 % trichloroacetic acid containing zoo fJ-g unlabeled L-histidine per ml. Samples were filtered on membrane filters and washed 4 times with equal volumes of 5 % trichloroacetic acid containing zoo fJ-g Lhistidine perml. The filters were placed in scintillation vials and dried for 30 min at 70°. Scintillation mix (composition: z,5-diphenyloxazole, 6.5 g; IA-bis-2-(4-methyl-5phenyloxazolyl)-benzene, 50 mg; naphthalene, 65 g; dioxane, 750 ml; xylene, ISO ml) Biochim, Biophys, Acta,

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was added and the samples counted in a Nuclear Chicago liquid scintillation counter Model 724. Measurement of [3-galactosidase The assay of {3-galactosidase has been described previously", using isopropyl-[3D-thiolgalactopyranoside (IPTG) as inducer and o-nitrophenyl-[3-n-galactopyranoside as substrate. Stability of inducer-specific repressor An examination of inducer-specific repression is greatly enhanced by the elimination of the masking effects of inducer-independent repression. Of several growth conditions tested, carbon restriction leads to the lowest degree of inducer-specific and inducer-independent repression, i.e., the highest differential rate of IJ-galactosidase synthesis", Unrestricted growth on glycerol leads to higher levels of inducerspecific and inducer-independent repression than does carbon-restricted growth. Unrestricted growth on succinate leads to a level of repression which is intermediate between those levels observed in carbon-restricted growth and unrestricted growth on glycerol (D. ] . CLARK, unpublished results). Succinate is therefore the preferred carbon source because it yields the lowest level of inducer-independent repression. In the presence of 7 . IO- 5 M IPTG, unr estricted succinate cultures form [3-galactosidase at approx. on e-t ent h the maximal rate. At the same concentration of IPTG, succinate-restricted cultures form [3-galactosidase at nearly one-half the maximal rate. The level of inducer-specific repressor for [3-galactosidase is probably lower in succinate-restricted cultures than in unrestricted (batch) cultures and ,B-galactosidase synthesis is therefore higher in succinate-restricted cultures at the same concentration of IPTG. The rate of repressor formation The rate of formation of repressor can be examined by observing the change in the differential rate of [3-galactosidase synthesis at sub-saturating concentrations of inducer by shifting from a condition with low repressor (succinat e-rest rict ed growth) to one with high repressor (succinate-unrestricted growth). The result of a shift from restricted growth to batch growth of a culture induced with sub-saturating concentrations of IPTG is shown in Figs . I and 2. The shift from succinate restriction to excess succinate is accompanied by a lag in growth of about 140 min (Fig. I). The differential rate of enzyme production in a succinate-restricted culture. induced with 7' 10- 5 M IPTG was 30· while the differential rate in the unrestricted culture at the same concent rati on of IPTG was 5.5· (Fig. 2). Before the shift, the differential rate in the experimental culture was coincident with the succinate-restricted control. Following the shift, initial repression of IJ-galactosidase occurred almost immediately but the steady-state rate of [3-galactosidase synthesis characteristic of the unrestricted culture is not attained until 1O0 min or 0.6 doublings after the shift.

• Expressed as enzyme units per cu lt ure.

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Fig . 1. Gr owt h of M L 3 during a sh ift from succinate-restricted growth t o succinate-unr estrict ed growth ,IS reflected by L-(14CJ his tidine in corp orat ion (. -e). X- X, growth of succinaterestricted growth; 0 - 0 , succinate-unrest ricte d growth . AU cu ltures were induced at an ab o sorbance of 0.250 with 7 ' 1 0 - 6 M J PTG in the presence of 2 flg L-[I"C]histidinejml which had a specific activity of 6.7 fl-Cfmg. Suc cinate was feel to the restricted culture at a growt h-limit ing rate overnight. The culture was then diluted to an absorbance of 0 .100 and slow feed continued to an absorbance of 0 . 2 5 0 at which time the culture was induced . The experimental culture (e -e ) was shifted at (A), 20 min after the addition of L-(HCJhistidine and IPTG, by the addition of succinate to a final concn. of 0 .5 'Yo . The unrestricted cont rol (0- 0) was ind uced after sev eral generations of exponential gro wth.

Rate of decay of f3-galactosidase repressor Repressor decay can be exam ined by following the change in the sub-maximal differential rate of tJ-galactosidase synthesis during the shift from unrestricted growth (high repressor) to succinate-restricted growth (low repressor) . A culture of ML 3 was induced with a sub-saturat ing concentration of IPTG during unrestricted growth and shifted to restricted growth (Figs. 3- 5). Ex cess succinate was exhausted in the shifted 80r

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Fig . 2. The change in differential synthesis of p-ga la ctosidase during a shift fr om succinaterestric te d growth to succinate-unrestricted growth (. -e ). (X -x) and (0- 0) show steadystate differential rate of ,a-galactos idase synthesis in a succinate-restricted culture and in an unrestricted-succinate cu lture, respectively. T he experimental culture (. -el was shifted from su ccinate-restrict ed growth t o unrestricted growth by the addit ion of 0.5 % succinate at (A).

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culture and growth became restricted at approx. 20 min after dilution as indicated by absorbance (Fig. 3). The shift from batch to restricted growth occurs between 14-20 min according to measurements of growth by incorporation of L- [14C]histidine (Fig. 4). The differential rate in the unrestricted control culture and in a culture that was shift ed from unrestricted growth to succinate-restricted growth are shown in Fig. 5. The differential rate of {1-galactosidase synthesis in the unrestricted control at 7 6

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Fig. 3. Gro wth of ML 3 (i +o+z+y-) during a sh ift from succinate-unrestricted growth to succinaterestricted growth as indi cated by absorbance us. time. At zer o t im e and exponential succinate cult ure of ML 3 was filtered and washed to remove exc ess succinate. The cells were resuspended with 200/lg succinatejml to allow growth to an absorbance of 0.550 . At an absorbance of 0.450 (A), the cells were diluted with medium lacking succinate and containing 2 /lg L-[14C]histidine/ml. The culture was allowed to equilibrate for 5 min at which time (E), IPTG was added to a final concn . of 7 ' 10-' M. After rapid mixing, the induced culture was divided. To the first part I mg succinatejml was added to maintain exponential growth (0-0). To the second part the slow addition of a feed solution of 5 mg euccinate/ml was started in a 500 ml volume at the rate of 1.5 ml/h which supported growth at approx, one-half of the exponential rate (e -e ). Fig. 4. .-e, growt h of ML 3 during shift from succinate-unrestricted gro wt h to succinaterestricted gr owt h as reflected by L-[14CJhist idine incorporation. 0-0. exponent ia l growth of unrestricted control. See Fig. 3 for ex periment al details.

7' IQ-5 M IPTG is 6*. Inducer was added at o. The arrow (A) indicates the shift from unrestricted to restricted growth as reflected by a change in the uptake of L-[14 C]histidine. The arrow (B) indicates the point at which the steady-state differential rate of ,a-galactosidase synthesis for restricted growth is attained. The interval A-B corresponds to approx. 50 min or to 0.3 doublings in the restricted culture.

Effect oj shift on constitutive synthesis oj {1-galactosidase The lag observed in Fig . 5 could be partially du e to the decay of inducer* Expressed as enzyme per

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Fig. 5. The differential rate of ,B-galactosidase formation in ML 3 as indi cated by the increase in enzy me us. increase in L-[uC] hist idine incorporation. (0 - 0 ,) the differential rate of the unrestricted control; (e-e). the differential rate of the experimental cult ur e shifted from unres tricted succinate to re stricted succ inate. Both cultures were induced with 7' IO-& M IPTG at o. The sh ift in gro wth as reflected by histidine incorporation is indicated by A. The point at which a. steady-state r ate of ,B-galactosidase formation occurs after the shift fr om hatch to restricted growth is indica t ed by B.

independent repressor which would mask the actual decay of inducer-specific repressor. A control experiment was done wit h ML 35. a permeaseless strain, constitutive for ,B-galactosidase. If the derepression observed in strain ML 3 during a shift is entirely due to the change in the level or the activity of the i-gene product, a shift from unrestricted to restricted growth with ML 35 should show no change in the differential rate of ,B-galactosidase formation. Fig. 6 shows the growth of ML 35 before and after the shift from batch to restricted growth. According to the increase in absorbance, the shift occurs at about go min after dilution. The shift to restricted growth occurs at about roo min as reflected by the incorporation of L-[14C]histidine (Fig. 7). The differential rate is 32 for both the unrestricted control and the shift to succinate-restricted growth. DISCUSSION

Carbon restriction leads to derepression of the synthesis of ,B-galaetosidase1o. Accordingly repressor should accumulate in a culture shifted from carbon-restricted growth to unrestricted growth. Such a shift is accompanied by rapid repression of ,B-galactosidase (Fig. 2). Repression is initiated rapidly but the steady-state is not attained for almost 0.6 doublings. Since carbon-restri ction derepresses,B-galactosidase synthesis, it could also derepress the enzymes involved in succinate metabolism. Exces s succinate added at the time ofthe shift could be degraded at a faster rate than normal causing momentary catabolite repression. Since catabolite repression consists of both inducer-specific and inducer-independent repression 9,the repression observed Bio chin«, B iopb ys , Acta, no (1965) 554-563

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Fig . 6. Growth of ML 35 (i-o+z+y-) during a shift from succinate-unrestricted growth to su ccinater estricted growth as indica te d b y absorbance us, t ime . At zero time an exponential succinate cu lture of ML 35 was filtered and washed to remove excess suc cinate. The cells were r esuspended with 200 Itg succinatejrnl t o allow growth to an absorbance of 0 . 5 5 0 . At an absorbance of 0.450 (A), the cells were diluted with medium lacking succinate and containing 2 fig L-[UClhistidine/m l. The culture was allowed to equilibrate for 5 min at wh ich time (E ) the culture was divided into 2 parts. To the fir st , I mg succinatejml was added to maintain exponential growth (0-0) . To the second , the slow addit ion of a feed solution of 5 mg succinatejml was started in a 500 ml volume at the rate of 1.5 rnl/h wh ich supported growth at approx. one-half of the exponential rate (e -. ). 8

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during the shift cannot be interpreted as an accurate indication of inducer-specific repressor formation . Shifts in temperature ar e subject to the same criticism ; temperature shocks may lead to a temporary increase in catabolite repress ion and thus mask the decay or formation of inducer-specific repressor-t. NOVICK8 did not consider catabolite effects during a shift in temperature but his main temperature shift was from 30-43° and in this range the level of inducer-independent repressor in strain ML 3 of E. coli seems to be constant!'. In 1964 , NOVICK indicated in a personal communication that the lag in enzyme synthesis after the temperature shift is decreased by the addition of inducer. It is therefore likely that NOVICK'S observation reflects the decay of inducer-specific repressor. Inducer-specific repressor is shown to be unstable (Fig. 5). Since the shift is from high repression (unrestricted growth) to low repression (succinate-restricted growth), the change in the differential rate of enzyme synthesis reflects the decay of repressor. Further, since succinate batch cultures have very low levels of inducerindependent repressor, the change in the differential rate of ,B-galactosidase synthesis probably reflects the decay of inducer-specific repressor. This shift (Fig . 5) was effected shortly after induction so the background of [14C]L-histidineand ,a-galactosidase would be low during the critical period of changing rate of enzyme synthesis. The dilution and induction, therefore, took place in a low concentration of su ccinate, which might restrict the growth rate prior to the shift. However, there is n o deviation from the unrestricted rate until zo min after induction (Figs. 3, 4)· Derepression of ,B-galactosidase occurs prior to the shift in histidine incorporation and shift in ab sorbance (Fig. 5). This is probably a result of depletion of succinate which affects the level of repressor before overall growth. Masking of inducer-specific repressor by inducer-independent repressor during a shift to succinate-restricted growth in ML 3 is eliminated by a comparison with the constitutive strain, ML 35. Inducer-specific repressor (i-gene product) is absent in the constitutive strain but inducer-independent repression can be pronouncedw.w, The shift with ML 35 (F igs. 6-8) was identical to the shift with ML 3 (Figs. 3-5) with the

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exception that IPTG was not necessary for induction. A comparison ofthe differential rates of ~-galactosidase synthesis in the inducible (Fig. 5) and in the constitutive strain (F ig. 8) after shifts to succinate-restricted growth, clearly indicates the derepression observed in ML 3 is unique to the inducible system. One therefore concludes that the increase in rate of ~-galactosidase synthesis reflects the decay of the i-gene repressor. The change in the differential rate (Fig. 5) occurs over a period of 50 min corresponding to 0.3 doublings of the succinate-restricted culture. Growth is, therefore, not essential for dilution of repressor. It seems quite reasonable on theoretical grounds to believe that inducer-specific repressor is a protein--". GALLANT' has provided evidence that one component for the control of alkaline phosphatase is protein and is unstable. On the other hand, inducerspecific repression is dependent upon the conditions of growth and is correlated with catabolite repression". Fig. 5 indicates that the inducer-specific repressor for tl-galactosidase is unstable. These results do not agree with those of N OVICK8 and of PARDEE l which indicate stability of i-gene product. The results of these experiments and those of N OVleK and PARDEE are, however, compatible with a two-component repression system. This view imagines an inactive stable protein ape-repressor which is activated by an unstable co-repressor. The co-repressor is sensitive to fluctuations in metabolic pools and is probably not a protein. This slight modification of the model of MONOD4 would explain how the repressor responds rapidly to metabolic shifts and simultaneously recognizes small molecules. NOTE ADDED IN PROOF

The interpretation that the changes in differential rate of tl-galaetosidase synthesis reflects changes in the concentration or activity of inducer-specific repressor assumes that the internal and external concentration of inducer are the same. Experiments by KEPES l 3 indicate that inducer concentration can be affected by activation of a pumping-out mechanism in glucose cultures of ML 3. It is unlikely, however, th at such findings relate directly to these experiments. If succinate cultures reflect a condition in which the internal concentration of inducer is equal to the external concentration of inducer then derepression can result either from an increase in internal inducer or from a decrease in activity or concentration of inducer-specific repressor. Since ML 3 lacks the permease for IPTG, there is no concentrating mechanism to activate, hence there is no means to produce a greater internal than external concentration of IPTG. It would therefore appear that derepression does not result from an increase in internal inducer concentration. Nevertheless, there is some doubt which can be eliminated only by actual measurements of internal inducer concentration. These experiments are in progress. Received September I3th, Ig6S

ACKNOWLEDGEMENT

I wish to acknowledge the support of this work by the Danish government and Biochim; BioPhys. Acta, IIO (19 6 5) 554-563

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the U.S. National Institutes of Health. In addition, I am extremely grateful to MAAL0E and G. EDLIN for many helpful comments during the preparation of this manuscript.

O.

REFERENCES I 2

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PARDEE, F . JACOB AND J . MONaD,]. M ol. Bioi.. I (1959) 165. A. B. PARDEE AND L. S. PRESTIDGE, B iochim. B iopbys . Acta, 49 (1961) 77. F. JACOB, R. SUSSMAN AND J . MONOD, Compt. Rend. , 254 (1962) 4<:14. J. MONOD, J . CHANGEUX AND F. JACOB, J. Mol . B iol., 6 (J963) 3 06. A . GAREN AND N . OTSUJI , J. Mol . Bioi., 8 (1964) 841. T. HORIUCHI AND A . NOVICK, Cold Spring Harbor Symp . Quant. B iol., <:6 (1961) 247· J. GALLANT AND R. STAPLETON, J. Mol. st«, 8 (1964) 442 . A. NOVICK, E. S . LENNOX AND F . JACOB, Cold Spring Harbor Symp . Quant. Biol., 28 (1963) 397· D. J. CLARK AND A. G, MARR, Biochim. Biopbys, Acta, 92 (1964) 85. D. J. CLARK AND A. G. MARR, Federation. Proc., 21 (1962) 235. A. G. MARR, J. L. INGRAHAM AND C. S. SQUIRES, J. Baoteriol., 87 (1963) 356. J. MANDELSTAM, Biochem , J ., 79 (1962) 489. A. KEPES, Biochim, Biopbys, Acta, 40 (1960) 70,

A. B .

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