COLI ON THE FORMATION- OF 3-GALACTOSIDASE

Transcription

1 JOURNAL OF BACTERIOLOGY Vol. 87, No. 2, p February, 1964 Copyright 1964 by the Anmerican Society for NMicrobiology Printed in U.S.A. EFFECT OF THE TEMPERATURE OF GROWTH OF ESCHERICHIA COLI ON THE FORMATION- OF 3-GALACTOSIDASE ALLEN G. MARR, JOHN L. INGRAHANM, AND CRAIG L. SQUIRES Department of Bacteriology, University of California, Davis, Calijornia ABSTRACT Received for publication 19 September 1963 IMARR, ALLEN G. (University of California, D)avis), JOHN L. INGRAHA'M, AND CRAIG L. SQUIRES. Effect of the temperature of growth of Escherichia coli on the formation of,3-galactosidase. J. Bacteriol. 87: The synthesis of,3- galactosidase was measured during exponential growth of Escherichia coli in a succinate-minimal medium over a temperature range of 1 to 43 C for the following: (i) a constitutive strain, and (ii) an inducible cryptic strain, induced maximallv with isopropyl-thio-f-n)-galactopyranoside (IPTG), or induced subinaxinially with IPTG. The differential rates of synthesis of 3-galactosidase were identical for the constitutive strain anid for the fully induced strain; the rates were constant from 2 to 43 C, and decreased progressively with a decrease in temperature below 2 C. Thus, in the absence of specific repression, the ability of E. coli to produce f-galactosidase decreases at low temperature. The differential rate of the submaximally induced culture was minimal between 2 and 3 C, and increased progressively with temperature both above 3 C and below 2 C. That the repressor concentration is maximal at 2 C was established by measuring the rate of induced synthesis of 3-galactosidase as a function of the concentration of IPTG; the relative concentrations of repressor were 1.:3.28:.25 at 4, 2, and 1 C, respectively. After an abrupt change in temperature, the differential rate of a submaximally induced culture changed gradually to the rate of the steady state, which is in agreement with the proposal that the effect of temperature is on the concentration of repressor and not on the equilibrium between riepressor and its site of action. The effect of temperature on catabolic repression was determined by comparing the differential rate of synthesis of,b-galactosidase by a constitutive strain grown in succinate-minimal medium with the rate in glucose-minimal medium at various temperatures; the ratio of the rates in the two media decreased progressively and approached 2. as the temperature of growth was increased. 356 Recently, ing, Ingraham, and M\Iarr (1962) suggested that the increase in the temperature characteristic of the rate of growth at low temperatures might result from a loss of regulation of certain repressible and inducible enzymes. Several exaamples of changes in regulation of the synthesis of enzymes with temperature of growth have been reported. Halpern (1961) found that glutamic decarboxylase of Escherichia coli is inducible at 37 C and partially constitutive at 3 C; Horiuchi and Novick (1961) isolated a mutant of E. coli which is inducible for 3-galactosidase at 14 C, but is constitutive at 43.5 C; Gallant (1962) isolated a mutant of E. coli which is constitutive for alkaline phosphatase above 39 C, but which becomes increasingly sensitive to repression by phosphate as the temperature of growth is decreased; Ng and Gartner (1963) showed that wild-type E. coli, which is inducible for tryptophanase at 3 C, cannot be induced to form tryptophanase below 15 C; and Maas (1961) reported a mutant of E. coli which synthesizes ornithine transcarbamylase at 37 C but not at 2 C. Several reports of an increase in nutritional requirements at low temperature may have a similar basis. For example, Campbell and Williams (1953) reported that the nutritional requirements of certain aerobic sporeformers are greater if the cultures are grown at low temperature. Mitchell and Houlahan (1946) isolated a mutant of Neurospora which had a greater requirement for riboflavine at 28 C than at 37 C. With the exception of the results of Halpern (1961), all of the above examples of changes in regulation with change in the temperature of growth indicate that repression is greater at low temperature. Horiuchi and Novick (1961) and Gallant (1962) assumed that the mutation to constitutivity at high temperature resulted from a loss of the thermostability of the repressor. An alternative explanation is that an increase in the

2 VOL. V-GALACTOSIDASE 87, 1964 FORMATION BY E. COLI.357 concentration of repressor at low temperatures of growth is characteristic of the wild type, and that mutants which are partially constitutive at high temperatures (i) make an altered repressor with less affinity for the receptor, or (ii) have an altered receptor with less affinity for the repressor. At low temperatures of cultivation, the level of repressor would be sufficiently high to maintain full repression; but, if the repressor level decreases at higher temperatures, this control would be lost. In this paper, we determined the effect of temperature of growth on the repression of f-galactosidase in strains of E. coli which produce the wild-type (i+) specific repressor of,b-galactosidase. MATERIALS AND METHODS Organisms and techniques of cultivation. E. coli strains ML3 (i+z+y-) and MIL35 (i-z+y-) were obtained from Jacques Monod. The cultures were grown in a basal salts medium (Marr, Nilson, and Clark, 1963) to which.5% sodium succinate,.2% glucose, or.4% glycerol was added as a carbon source. The cultures in tubes (3 by 3 cm) were immersed in a water bath at the stated temperature i.5 C, and were aerated by sparging sterile air through an 8-mm alundum disc submerged in the culture. Growth was followed by periodic measurement of optical density in a 1-cm cell at 6 my with a Beckman model DU spectrophotometer, and with the geometry specified by Marr et al. (1963). The precision of individual measurements of optical density (OD) is <.7% (SD) from.1 to.4 OD, the range in these experiments. The OD is 1.97 per mg (dry weight) of cells per ml, and is linear 4.6% to.3 OD and i1% to.4 OD. Induction of f-galactosidase. Cultures were grown for several generations in the medium containing the particular carbon source, and for at least two generations of exponential growth at the temperature at which the experiment was to be made. After the culture reached approximately.3 OD, 5 ml of the culture were transferred to 15 ml of medium at the same temperature and containing a sufficient concentration of inducer, isopropyl-f3-d-thiogalactopyranoside (IPTG), to give the appropriate final concentration. At intervals, samples were withdrawn and added to tubes containing chloramphenicol to give a final concentration of 5,ug/ml. These tubes were held at C prior to the assay for f-galactosidase. In some experiments, the chloramphenicol was omitted, and samples of the culture were immediately treated with toluene as described below. Assay of f3-galactosidase. With a vortex mixer, 1 ml of sample and.1 ml of toluene were emulsified. After the toluene was evaporated at 37 C, x ml of sample,.8 - x ml of basal medium, and.2 ml of.133 m o-nitrophenyl- 3-D-galactopyranoside (GNPG) in.25 Mi sodium phosphate buffer (ph 7.) were allowed to react at 28 C until a yellow color developed (such that the final absorbancy was less than.8). The reaction was stopped by the addition of.5 ml of 1 M Na2CO3. The o-nitrophenol produced was computed from the absorbancy at 42 m,u, assuming a molar absorbancy of 4,75 cm-'. One unit of,f-galactosidase was taken as the amount of enzyme required to hydrolyze 1 muamole of ONPG per min under the conditions of the assay (Herzenberg, 1959). The precision of replicate assays of samples containing 7.66 units was i4.7% SD. Determination of differential rates. The differential rate of synthesis of f-galactosidase was determined as the slope of a plot of the units of enzyme per ml of culture, as a function of the OD. Each rate is based on measurement of eight or more samples of a culture taken lperiodically during exponential growth over an interval of at least 1.5 generations. After induction at low temperature ( <25 C), differential rate increases for approximately one generation, after which the rate is constant; the final constant value is reported in the results. RESULTS Effect of temperature of growth on the maximal rate of synthesis of 3-galactosidase. The effect of temperature on the synthesis of f-galact.osidase in the absence of specific repression was estimated by measuring the differential rates of both a constitutive strain, E. coli ML35, and an inducible strain, E. coli ML3, which was induced maximally by 7 X 1-4 M IPTG (Fig. 1, curve A). Both the constitutive strain and the maximally induced, inducible strain gave identical results. The differential rate is constant over the temperature range of 2 to 43 C. Below 2 C the

3 358 MARR, INGRAHAM, AND SQUIRES J. BACTERIOL. Jcc z 111 LL. Of I ~~~~~ -2/ / o. II -1 / _ e~~~~~~~ C _- I I TEMPERATURE FIG. 1. Effect of the temperature of growth on the synthesis of -galactosidase by a constitutive strain, Escherichia coli MLS5 (), and by an inducible strain, E. coli MLS, maximally induced by 7 X 1-4 M IPTG (), and submaximally induced by 5 X 1-5 M IPTG (e). The cultures were grown in succinate-minimal medium. The ordinate is the differential rate (units of f3-galactosidase per ml per OD unit). rate decreases progressively, and at 1 C the rate is only half the rate at 2 C. Effect of the temperature of growth on repressor concentration. The effect of temperature on the concentration of repressor was estimated by measuring the synthesis of #-galactosidase by a cryptic strain (ML3) induced with 5 X 1-5 M lptg, a submaximal concentration of inducer (Fig. 1, curve B). The differential rate is minimal at about 2 C, and increases at both higher and lower temperatures of growth. If the inducer reacts with repressor either directly or indirectly, submaximal induction must represent a condition in which only a fraction of the total repressor is inactivated. Under this condition, the differential rate of enzyme synthesis should be inversely related to the B amount of active repressor in the cell. Hence, if the concentration of inducer is constant, differential rate should be inversely related to the concentration of repressor. With this assumption, the results indicate that the concentration of repressor of f3-galactosidase is maximal in cells grown at 2 C, and decreases at both higher and lower temperatures of growth. The conclusion that the concentration of repressor was maximal at 2 C was verified by measuring the differential rate of synthesis of,b-galactosidase as a function of the concentration of inducer at 1, 2, and 4 C. The results were analyzed according to the following equation (Marr and Marcus, 1962): 1 K pp= p (1-2) + p p max Pmax (1) in which P is the differential rate at any concentration of inducer, I; Pmax is the limit of P as I approaches infinity; and K is a parameter linearly related to the concentration of repressor. The specific assumptions on which this equation is based need not concern us here; a similar form was developed from different assumptions by Roberts, Britten, and McClure (1961). That equation 1 adequately describes the relationship between differential rate of synthesis of,b-galactosidase and the concentration of inducer was demonstrated by Boezi and Cowie (1961), by the analysis of Marr and Marcus (1962) of the data of Herzenberg (1959), and by Clark (1963). The results, analyzed graphically according to equation 1, are shown in Fig. 2a. The slope and ordinate intercept were determined by regression of the ordinate on the abscissa. The value of K was computed as the quotient of the slope and intercept with the following results: (i) K4 c =.98 X 1-8; (ii) K2C = 3.27 X 1-8, and (iii) Koc =.267 X 1-8 mole2 X liter-2. Because this analysis heavily weights the values at low concentrations of inducer, the analysis was repeated with the following transformation (Hofstee, 1952): P = -KQ2) +Pmax (2) The graphical analysis of the results according to equation 2 are shown in Fig. 2b. The slopes were determined by regression with the following results: (i) K4C = 1.14 X 1-8; (ii) K2C =

4 V1-GALACTOSIDASE FORMATION BY E.COL3 VOL. 87, I-2 X (P/I2)x 1-' FIG. 2a. Effect of the concentration of inducer on the differential rate of,b-galactosidase synthesis by a cryptic strain, Escherichia coli ML3, grown at 1, 2, and 4 C in succinate-minimal medium. P is the differential rate, and I is the molar concentration of IPTG. Data were analyzed by regression according to equation 1 (see text) with the following results: 4 C, K = X 18, Pmax = 3,57 i: 44; 2 C, K = 3.68 i.12 X 1-8, Pmax = 3, ; 1 C, K =.273 ±.19 X 1-8, Pmax = 1, FIG. 2b. Data of Fig. 2a analyzed by regression according to equation 2 (see text) with the following results: 4 C, K =.98 i4.14 X 1-8, Pmax = 3,46 ik 1,23; 2 C, K = 3.27 ±.21 X 1-8, Pmax = 3,2 ± 6; 1 C, K =.265 i.1x 1-8, Pmax = 1,162 i X 1-8, and (iii) Klo c =.273 X 1-1 mole2 X liter-2. The results of the two analyses are in good agreement (the per cent difference is 15.3, 11.8, and 2.2, respectively), which confirms both the linearity of the functions and the estimate of K. With the assumption that K is a linear function of the concentration of repressor, the relative concentrations of repressor are 1.:3.28:.25 at 4, 2, and 1 C, respectively. The values of Pmax at 2 and 4 C are not significantly different; however, at 1 C, Pmax is significantly diminished, confirming previous conclusions from the results in Fig. 1. Effect of an abrupt change in temperature. The rate of change, after a change in temperature, of the rate of synthesis of,b-galactosidase by strain ML3 induced submaximally provides additional evidence that the temperature of growth affects the concentration of repressor. If the previous observation were primarily a result of the effect of temperature on an equilibrium, a change in the temperature of growth would give an immediate change in the differential rate. However, if the observations are dependent on a change in the concentration of repressor, a change in the temperature of growth would be followed by a transient differential rate. The effect of abrupt changes in temperature is shown in Fig. 3. After an increase in temperature from 25 to 37 C, the differential rate gradually increases until the rate of the control at 37 C is attained. Likewise, after a decrease in temperature from 37 to 25 C, the differential rate gradually decreases until the rate of the control at 25 C is attained. The transient rates were analyzed by constructing the function corresponding to the rate of the control for the postshift temperature (dotted lines in Fig. 3), by determining the deviates of the experimental points from this function, and by plotting the logarithms of the deviates as a function of the time after the change in temperature (Fig. 4). If the transient is an exponential function of time, this analysis should be linear.

5 36 MARR, INGRAHAM, AND SQUIRES J. BACTERIOL. c: U) : (I) (-) n-j (-5 D -8-6 /D 7 C) -4 37C 25-37C _ * / 2~~~~~~~ -2 X/,' a 1-J LLJ U) C/) I (D I / I *.2.3 Of OPTICAL DENSITY OPTICAL DENSITY '7/,' /S,-,, 'IC'.2.3 I II b -4 _ -3 / 2-37-* 25C 25C ', ' -2,- FIG. 3. Effect of an abrupt change in temperature on the synthesis of j-galactosidase by a cryptic strain, Escherichia coli MLS, submaximally induced by 6 X 1- M IPTG. The cultures were grown in glycerolminimal medium. (a) Shift from 25 to 37 C; (b) shift from 37 to 25 C. / '/ / I / I / The response time was computed from the slopes of the functions in Fig. 4. The response time of the transient after the change from 37 to 25 C was.29 generations. The response time for the transient after the change from 25 to 37 C was.45 generations. The results of this experiment confirm the hypothesis that the concentration of repressor is a function of the temperature of growth. For comparison, a similar analysis of the data of Horiuchi and Novick (1961; Fig. 1) for the transient rate after a shift of the thermal constitutive from 45 to 14 C, which has been interpreted as the time required for the concentration of repressor to increase, gives a response time of.54 generations. Effect of temperature on catabolic repression. Catabolic repression (iiagasanik, 1961) was estimated by comparing the differential rate of enzyme synthesis by a culture of constitutive strain MIL35 growing in suceinate-minimal medium with the rate in glucose-minimal over the temperature range of 1 to 4 C (Fig. 5). The ratio of the differential rate in succinate-minimal mediurn to the rate in glucose-minimal medium was taken as an index of catabolic repression. The ratio is 5.8 at 1 C, and decreases progressively as the temperature of growth is increased, indicating that catabolic repression increases at low temperature. The ratio is 2. from 3 to 4 C, in agreement with the results of Cohn and Horibata (1959) and of Brown (1961). DISCUSSION The temperature of growth has two major effects on the synthesis of 3-galactosidase. One effect, the progressive decline in the rate of synthesis either by a constitutive strain or by a fully induced, inducible strain, cannot be mediated by a change in specific repressor. This effect could reflect an increase in catabolic repression by the carbon source, suceinate, at low temperature, or it may result from an effect of temperature directly on the expression (transcription) of the structural gene. The effect of temperature on constitutive or on maximally induced synthesis of f-galactosidase is apparently similar to the effect of temperature on the synthesis of tryptophanase. Ng (1963) found that wild-type E. coli produces decreasing amounts of tryptophanase as the temperature of growth is reduced, and cannot form trypto-

6 VOL. 87, 8-GALACTOSIDASE 1964 FORMATION BY E. COLI 361 phanase at less than 15 C. Mutants which do form tryptophanase inducibly at low temperature were isolated (Ng and Gartner, 1963). Gartner and Riley (personal communication) found that this mutational locus is closely linked to the structural gene, and is probably an operator. The failure of the wild-type to produce tryptophanase at low temperature presumably results from a failure of the operator to function at low temperature. That the temperature of growth strongly affects the concentration of specific repressor is suggested by the change with the temperature of growth of the rate of synthesis of f-galactosidase by a submaximally induced, cryptic strain; this is confirmed by titrations with inducer and by the transient rate of synthesis observed after a change in the temperature of growth. The concentration of repressor was found to be maximal near 2 C, and decreased markedly at 4 and 1 C. The low concentration of repressor at 1 C may explain the observation of Ng et al. (1962) LOG,(AE/ML) ;~~~ \ ~~ -5 14G (Data of Horiuchi & )\ \ \ ~~~~~~~Novick) 25 b37c\ _ \ o\ \ *7 \ o I.5 GENERATIONS AFTER SHIFT FIG. 4. Analysis of the transient rates after a change in temperature. The logarithms of the deviates of the experimental points from the dotted lines in Fig. 3 are plotted as the ordinate as a function of the time in generations after the change in temperature. A similar analysis was made of the data of Horiuchi and Novick (1961). The curves were fitted by regression. The response times were calculated as.434/slope. LLJ z LLJ L EL 2 - / - * o -1 / 1O C I g-i I I TEMPERATURE FIG. 5. Effect of temperature of growth on the synthesis of fi-galactosidase by a constitutive strain, Escherichia coli MLS5, growing in glucose-minimal () or in succinate-minimal () medium. The ordinate is the differential rate (units of,3-galactosidase per ml per OD unit). that the repression by glucose of the induced synthesis of,b-galactosidase by E. coli ML3 (i+z+y+) is relieved at low temperature, a result which apparently is contradicted by our finding that repression by glucose increases at low temperature. More rapid induction of galactoside permease in y+-strains at low temperature would be permitted by the low concentration of repressor. This, coupled with the greater accumulation of galactosides at low temperature (Kepes, 196), would lead to rapid establishment of the maximal differential rate of synthesis of,b-galactosidase. This proposal is confirmed by the fact that the kinetics of induction of E. coli ML 3 growing at high temperature in glucose-minimal medium resemble the "autocatalytic" kinetics reported by Novick and Weiner (1957) for induction of y+-strains by limiting concentrations of inducer, but, at low temperature, the differential rate is not a function of time after induction

7 ,362 MARR, INGRAHAM, AND SQUJIRES J. BACTERIOL. (Ng, 1963). Thus, if a comparison is made before the maximal rate is established at high temperature, it would appear that the catabolic repression is greater at high temperature. The response times of the transient rates of submaximally induced synthesis of,b-galactosidase after a change in temperature of growth are comparable to the response time computed from the data of Horiuchi and Novick (1961) for a thermal constitutive shifted from high to low temperature. The similarity suggests that the transient rates have the same basis; namely, a change in concentration of repressor. That the response time is less than one generation indicates that repressor is stable neither in the thermal constitutive nor in the wild type. The fact that in wild-type (i+) strains the concentration of repressor decreases at temperatures near the maximum for growth reopens the question of the basis of thermal constitutivity (see introduction). The repressor in such mutants need not be uniquely thermolabile; a decreased amount of repressor, a change in the affinity of the repressor for the operator, or a decrease in the affinity of the operator for repressor, together with the normal variation in concentration of repressor with temperature, could explain thermal constitutivity. ACKNOWLEDGMENT This work was sup)p)orted by grants-in-aid fromi the National Science Foundation and AJ from the National Institutes of Health, U.S. Public Health Service. LITERATURE CITED BOEZI, J. A., AND 1). B. COWIE Kinetics studies of,b-galactosidase induction. Biophys. J. 1: BROWN, 1). 1) Catabolite repression. Cold Spring Harbor Symp. Quant. Biol. 26: C VAIPBELL, L. L., JR., ANI). B. WILLIAMS The effect of temperature on the nutritional requiremenit of facultative and obligate ther mophilic hacteria. J. Bacteriol. 65: CLARK, 1). J Catabolite repression of - galactosidase in Escherichia coli. Ph.D. Thesis, IUniversity of California, Davis. COHN, AI., AND K. HORIBATA Physiology of the inhibition by glucose of the induced synthesis of the f-galactosidase-enzyme system of Escherichia coli. J. Bacteriol. 78: GALLANT, J. A Thermal derepression of alkaline phosphatase synthesis. Biochem. Biophys. Res. Commun. 8: HALPERN, Y. S Temperature-dependent inducer requirement for the synthesis of glutamic decarboxylase by Escherichia coli. Biochem. Biophys. Res. Commun. 6: HERZENBERG, L. A Studies on the induction of f-galactosidase in a cryptic strain of Escherichia coli. Biochim. Biophys. Acta 31: HOFSTEE, B. H. J On the evaluation of the constants Vm and Km in enzyme reactions. Science 116: HORIUCHI, T., AND A. NOVICK A thermolabile repression system. Cold Spring Harbor Symp. Quant. Biol. 26: KAPEs, A Etudes cinetiques sur la galactoside-permease d'escherichia coli. Biochim. Biophys. Acta 4:7-84. MAAS, W. K Studies on repression of arginine biosynthesis in Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 26: MAGASANIK, B Catabolite repression. Cold Spring Harbor Symp. Quant. Biol. 26: MARR, A. G., AND L. MIARCUS Kinetics of induction of mannitol dehydrogenase in Azotobacter agilis. Biochim. Biophys. Acta 64 : MARR, A. G., E. H. NILSON, AND D. J. CLARK The maintenance requirement of Escherichia coli. Ann. N.Y. Acad. Sci. 12: MITCHELL, H. K., AND M. B. HOULAHAN Neurospora. IV. A temperature-sensitive, riboflavinless mutant. Am. J. Botany 33: NG, H The physiological basis for the existence of a miilinial growth temperature in Escherichia coli. Ph.D. Thesis, University of California, T)avis. NG) H., AND T. K. GARTNER Selection of mutants of Escher-ichia coli constitutive for tryptophanase. J. Bacteriol. 85: NG, H., J. L. INGRAHAM, AND A. G. MARR DIamage and derepression in Escherichia coli resulting from growth at low temperatures. J. Bacteriol. 84: NOVICK, A., AND M. WEINER Enzyme induction as an all-or-none phenomenon. Proc. Natl. Acad. Sci. U.S. 43: ROBERTS, R. B., R. J. BRITTEN, AND F. T. Mc- CLURE A model for the mechanism of enzyme induction. Biophys. J. 1: