:~olec. Gen. Genetics 106, 371--377 (1970)

Thermosensitive Regulation of D-Serine Deaminase Synthesis in a Mutant of Escherichia coli K 12 ELIZABETHM c F . ~ and MA~Ii C. t t ~ c z Department of Microbiology, New York University School of Medicine ~qew York, New York 10016, U.S.A. Received January 23, 1970 Summary. A mutant strain of Escherichia coli K12 is described, which exhibits thermosensitive regulation of D-serine deaminase synthesis. The mutant is distinct in its physiological properties from iTL and i Tss mutants of the lac systems, although it has elements of

similarity with both. A model is presented to explain its properties. Introduction

Mutations to temperature-sensitive regulation of enzyme formation have been well characterized for several inducible enzyme systems in bacteria (Sadler and Novick, 1965; Gallant and Stapleton, 1963). The thermosensitivity in these systems has been traced to two types of defective regulatory gene products (repressors). In one type, denoted i TL by Sadler and Novick (1965), the repressor functions fairly well at low temperature but becomes progressively more unstable with increasing temperature of growth. In the other, iTSS, the finished repressor functions fairly well at all temperatures, but its synthesis is temperature sensitive, and at high temperature little or no active product is formed. The lac repressor is a polymeric protein, and it is thought that the iTSS mutation affects the stability of the monomers much more than that of the final polymer. In accord with this theory, the temperature range for the transition from inducibility to constitutivity is much narrower in iTSS mutants than in i TL mutants (Sadler and Novick, 1965). In both cases enzyme formation is inducible at low temperature; constitutive at high temperature (42 ° C). We have previously described a mutation, dsdC1, which results in temperaturesensitive regulation of D-serine deaminase synthesis (MeFall, 1964). Physiologically, as we report below, it has elements of similarity to both the i TL and iTSS mutations of the lactose system. Materials and Methods Strains, media, methods of cultivation, and D-serine deaminase assay have been described previously (McFall, 1967a). Cells were always grown in minimal medium, with glycerol as sole carbon source. Q~C)L-arginine uniformly labelled, specific activity 220 ~c/i~M, was purchased from the New England Nuclear Corporation. For incorporation studies, minimal medium was supplemented with 50 ~zg/ml (14C)L-arginine, specific activity 0.22 ~c/~M. To determine growth rates QaC)L-arginine uptake into protein was measured by the method of Roodyn and Mandel (1960). Bacterial growth was also sometimes measured as increase in optical density at 660 m~ in a Zeiss spectrophotometer (O.D. 1.5~109 cells/ml). ~Nalidixic acid (Goss, Dietz and Cooke, 1965) was kindly provided by Dr. Harold Anderson of Winthrop Laboratories. 25*

372

E. McFall and M. C. Heinez: 20-

\ 18 8

u

-,,,,, -\

.,.,,

-

Q. U3

3.1

I 3.2

I &3 I/TxlO 3

I 3/-,

I 3.5

Fig. 1. Effect of temperature on rate of synthesis of D-serine deaminase. Cells were grown in minimal medium for at least 10 generations at temperatures from 20 to 43° C. Rates of enzyme synthesis are presented as steady state specific activities at the various temperatures

Results

Temperature Dependence o/D-Serine Deaminase Synthesis in EM 1100 The basal level of D-serine deaminase in wild type E. coli K12 is about 1/1000 that found in fully induced or fully constitutive cells (MeFall, 1964). Fig. 1 illustrates the temperature dependence of constitutive D-serine deaminase synthesis in the m u t a n t strain EM 1100 (dsdCl). The data are plotted in terms of log specific activity vs. 1/T. The enzyme formed by the dsdCl m u t a n t has been shown to be completely stable in this temperature range (MeFall, 1964). The rate of enzyme formation at 20 ° C, the lowest temperature tested, is about 40 x that of the basal rate in the inducible wild type ; the rate at 43 ° C is about 600 × that of the basal rate (McFall, 1964). The Q10 for this transition from inducibility to eonstitutivity is 2--3, about what might be expected for a simple organic reaction (rather than a protein denaturation). This suggests that the dsdC1 product (repressor) is not of t h e / a c iTSS type. Since the primary effect of temperature in the iTSS type of m u t a n t is at the level of subunits rather than the finished product, one sees a much sharper thermal transition from inducibility to constitutivity.

E//ect ol Temperature Shi/t I n the experiment of Fig. 1, cells were grown for long periods at the various ~emperatures, to establish steady-state specific activities. To determine the immediate effect of temperature variation on enzyme synthesis - - and thus by inference on the repressor - - we performed a series of experiments involving rapid shifts from one temperature of growth to another. I n the first experiment, shown in Fig. 2, a culture of E~¢I 1100 was grown at 24 ° C, centrifuged, washed, resuspended in fresh medium lacking glycerol, and divided into three equal portions, A, B, and C. Glycerol was added to cultures A and B. Growth of culture A, the control, was continued at 24 ° C; cultures B and C were grown at 42 ° C for one hour, then glycerol was added to culture C and both cultures were returned to 24 ° C. Samples were taken as indicated for measure-

Thermosensitive D-Serine Deaminase Synthesis

373

o/ / °

10 -

o

o/

£ o

0

~/4) I

I

I

I

I

1

2

3

h

5

Arginine CIt• incorporation/ml x10 z'

Fig. 2. Effect of shift from 24 to 42 ° C, and subsequent shift from 42 to 24° C, on growth and synthesis of D-serine deaminase. Cells were grown in minimal medium supplemented with 50 ~g/ml L-arginine to a density of 2 × 10S/ml, then centrifuged, washed, resuspended in minimal medium lacking glycerol and supplemented with 50 tzg/ml (14C)L-arginine, specific activity 0.35 tzc/~M, and divided into three equal portions. Glycerol was added to cultures A (A--A) and B (o--o), not to C (o--o). Culture A, the control, was grown ~t 24°C for the remainder of the experiment. Cultures B and C were grown at 42 ° C for one hour, then glycerol was added to culture C and both were returned to 24°C (arrows). Samples were taken at 15 rain intervals throughout the experiment for measurement of enzyme activity and of growth in terms of (14C)L-arginine uptake

m e n t of growth, m e a s u r e d b y i n c o r p o r a t i o n of (14C)L-arginine a n d e n z y m e activ i t y . I t m a y be seen t h a t culture C, which was h e a t e d a t 42 ° C in t h e absence of a c a r b o n source, d i d n o t grow significantly d u r i n g t h e p e r i o d of heating, a n d t h a t g r o w t h r e s u m e d on its r e t u r n to 24 ° C, when i t received glycerol. T h r o u g h o u t t h e e x p e r i m e n t , however, t h e differential r a t e of e n z y m e s y n t h e s i s in this c u l t u r e was t h e s a m e as in t h e control, A. T h u s s i m p l y h e a t i n g a t 42 ° C was n o t sufficient to cause a n increase in t h e r a t e of e n z y m e synthesis. I n culture B, t h e r e was a s i g n i f i c a n t lag a f t e r t h e shift t o 42 ° C u n t i l t h e r a t e of e n z y m e s y n t h e s i s increased, a n d a n o t h e r lag a f t e r t h e shift d o w n to 24 ° C u n t i l this r a t e declined. These lags are shown in m o r e d e t a i l in Figs. 3 a n d 4, which p r e s e n t shift u p a n d shift d o w n e x p e r i m e n t s in which s a m p l e s were t a k e n m o r e f r e q u e n t l y . I t m a y be seen t h a t while t h e lag a f t e r shift u p t o 42 ° C is significant, i t is o n l y a fraction, a b o u t one-sixth, of t h e g e n e r a t i o n time. This also argues a g a i n s t t h e p o s s i b i l i t y t h a t dsdCl m i g h t be a lac iTSS t y p e of m u t a t i o n . Such m u t a n t s requh'e more

374

E. i~IcFall and M. C. Heincz:

/ 13t~

11

E

IC

/

/

.c_

D

/ k

0.5

1.0 Optical density Fig. 3

1.5

0.7

0.8 Optical density

I

0.9

Fig. 4

Fig. 3. Effect of shift from 24 to 42 ° G on rate of D-serine deaminase synthesis. Cells were grown at 24 ° C to about 4 × 10S/m] in the presence of glycerol, then shifted to 42 ° C. Samples were taken for measurement of enzyme activity and of growth in terms of optical density at 10, 20, and 30 rain before the shift (arrow) and at 0, 5, 10, 20, 30, 45, and 60 min afterwards Fig. 4. Effect of shift from 42 to 24 ° C on rate of D-serine deaminase synthesis. Cells were grown at 42 ° C to cell density about 5 × 10S/ml, then shifted to 24° C. Samples were taken for measurement of enzyme activity and optical density at 10 min intervals for 90 rain following the shift (arrow)

t h a n a g e n e r a t i o n before d e r e p r e s s i o n in response to a t e m p e r a t u r e shift is o b s e r v e d (Sadler a n d N o v i e k , 1965). T h e r e is, however, a n a p p a r e n t c o n t r a d i c t i o n in t h e results of t h i s section. T h e d a t a of curve B, Fig. 2 a n d Figs. 3 a n d 4 w o u l d be u n d e r s t a n d a b l e if dsdC1 specified f o r m a t i o n of a t h e r m o l a b f l e repressor of t h e i TL t y p e . T h e y suggest t h a t a brief p e r i o d a t 42 ° C is n e c e s s a r y for i n a c t i v a t i o n of t h e dsdC1 p r o d u c t . U p o n r e t u r n to 24 ° C, some considerable p e r i o d of g r o w t h is n e c e s s a r y for synthesis of sufficient n e w r e p r e s s o r to depress t h e r a t e of e n z y m e synthesis. H o w e v e r , t h e d a t a f r o m c u l t u r e C of Fig. 2 do n o t agree w i t h t h i s i n t e r p r e t a t i o n . A l t h o u g h culture C was h e a t e d for a considerable time, no loss of f u n c t i o n of t h e repressor was observed. These results, t a k e n t o g e t h e r , i n d i c a t e some f u n c t i o n of g r o w t h or e n e r g y m e t a b o l i s m is n e c e s s a r y for i n a c t i v a t i o n .

E//ect o/Nadalixic Acid on D-Serine Deaminase Synthesis after Temperature Shi/t W i t h lac i Tss m u t a n t s , B a r b o u r , Gross a n d N o v i c k (1968) o b s e r v e d t h a t b y blocking D N A s y n t h e s i s w i t h nalidixic a c i d a f t e r a shift f r o m 24 t o 42 ° C, t h e y p r e v e n t e d a n increase in fl-galactosidase synthesis. T h e y c o n c l u d e d t h a t t h e i Tss p r o d u c t is n o t g r o w t h u n s t a b l e , as p r e v i o u s l y s u g g e s t e d ( G a l l a n t a n d S t a p l e t o n , 1963), b u t t h a n a n " e s c a p e " s y n t h e s i s occurs a t 42 ° C, as t h e n u m b e r of lac o p e r a t o r s increases while t h e a m o u n t of repressor r e m a i n s c o n s t a n t .

Thermosensitive D-Serine Deaminase Synthesis 20

1/3

"E:

375

/

/

o

o IC - -

-~zx /

o

/

E o -8

m c

"~- 5 .o i

E3

1

2

3

Z,

5

6

Arginine C14 incorpomtion/ml xl04

Fig. 5. Effect of nalidixic acid on the rate of D-serine deaminase synthesis after a shift from 24 to 42 ° C. Cells were grown to a density of about 2 × 10S/ml in minimal medium supplemented with 50 ~g/ml L-arginine. (laC)L-arginine was added to give a final specific activity of 0.35 t ~ c / ~ , and the culture was divided into three equal portions, A (o--.), B (©--o) and C (/X--/X). Cultures A and B received nalidixic acid, final concentration 20 ~g/ml, and all three cultures were shifted to 42 ° C. 60 min after the shift (arrows) cultures ]3 and C were returned to 24 ° C. Samples were taken at 10 rain intervals throughout the experiment for measurement of D-serine deaminase activity and of growth in terms of (14C)L-arginine uptake

A c c o r d i n g l y , we p e r f o r m e d a n e x p e r i m e n t similar to t h a t of B a r b o u r , Gross a n d N o v i c k (1968). A c u l t u r e of E M 1100 was first g r o w n a t 24 ° C, t h e n d i v i d e d i n t o t h r e e equal portions. N a l i d i x i e a c i d was a d d e d to t w o (A a n d B) a n d all were shifted to 42 ° C. A f t e r one hour, culture A a n d t h e control culture, C, were r e t u r n e d to 24 ° C, c u l t u r e B was left a t 42 ° C. T h e results are p r e s e n t e d in Fig. 5. A l t h o u g h D N A synthesis in cultures A a n d B was 95 % i n h i b i t e d b y nalidixic acid, i t is clear t h a t t h e r a t e of D-serine d e a m i n a s e synthesis i n c r e a s e d a t t h e s a m e t i m e a n d to t h e s a m e e x t e n t as in c u l t u r e C. Moreover, synthesis c o n t i n u e d a t t h e s a m e differential r a t e in all cultures for t h e d u r a t i o n of t h e e x p e r i m e n t , a l t h o u g h t h e r a t e of p r o t e i n synthesis was s o m e w h a t lower in t h e nalidixic acid cultures t h a n in t h e n o r m a l one. W e conclude t h a t t h e a p p a r e n t g r o w t h d e p e n d ence of escape s y n t h e s i s in E M 1100 is n o t a f u n c t i o n of D N A synthesis.

The E//eet o/ Varying Time o/ Growth at 42 ° C on the Subsequent Rate o / E n z y m e Synthesis at 24 ° C I f t h e r e is a g r o w t h r e q u i r e m e n t for i n a c t i v a t i o n of t h e repressor a t 42 ° C~ one should f i n d t h a t shortening t h e p e r i o d of e x p o s u r e to t h e high t e m p e r a t u r e results in a r e ] a t i v e l y s h o r t e r lag before t h e r a t e of e n z y m e s y n t h e s i s declines. A c c o r d i n g l y , we t r a n s f e r r e d p o r t i o n s of a c u l t u r e p r e g r o w n a t 24 ° C to 42 ° C for p e r i o d s of 30, 45, a n d 60 rain, t h e n r e t u r n e d t h e m t o 24 ° C. T h e resu]ts are pres e n t e d in Fig. 6. I t is clear t h a t t h e s h o r t e r t h e e x p o s u r e t o 42 ° C, t h e m o r e r a p i d is t h e decline in t h e r a t e of e n z y m e synthesis a t 24 ° C.

376

E. MeFall and M. C. Heincz:

/

hC

A

Zk

3O

y

.~ 20 E

._~ i

o 10

J l

I I I I 2 h 6 8, I0 A'rginine C1~ incorporation/ml xl0 a

Fig. 6. Effect of variation in time of exposure to 42 ° C on subsequent rate of D-serine deaminase synthesis. Cells were grown at 24° C in minimal medium supplemented with 50 ~g/ml L-arginine to about 2 × 10S/ml. (laC)L-Arginine was then added to give final specific activity of 0.35 ~c/ [zM, the culture was divided into three equal portions, A (© ¢), B (©--©) and C (A--A), and all three portions were shifted to 42 ° C. At 30 rain culture A was returned to 24 ° C, at 45 min, culture B, and 60 min, culture C. Samples were taken at 15 min intervals for measurement of enzyme activity and of growth in terms of (14C)L-arginine uptake

Discussion Several o b s e r v a t i o n s are c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t t h e dsdCl m u t a t i o n is of t h e iTL class, t h a t is, t h a t dsdCl specifies a r e g u l a t o r y s u b s t a n c e (represser) which is t h e r m o l a b i l e . T h e t i m e r e q u i r e d for t h e r m a l derepression is a s m a l l f r a c t i o n of a generation. T h e kinetics of t h e r m a l derepression a n d subs e q u e n t r e c o v e r y are t h e s a m e in t h e presence a n d absence of sufficient nalidixie a c i d to p r e v e n t D N A synthesis, suggesting t h a t derepression is n o t due to d i l u t i o n of s t a b l e r e p r e s s e r molecules as D-serine d e a m i n a s e o p e r a t o r s m u l t i p l y . The longer a c u l t u r e grows a t high t e m p e r a t u r e (42 ° C), t h e m o r e slowly does t h e r a t e of e n z y m e synthesis decline u p o n a r e t u r n to a low g r o w t h t e m p e r a t u r e (24 ° C). H o w e v e r , m e r e h e a t i n g a t 42 ° C, in t h e absence of growth, does n o t result in derepression. I f t h e r e p r e s s e r were r a p i d l y a n d i r r e v e r s i b l y i n a c t i v a t e d a t 42 ° C, one w o u l d e x p e c t a b u r s t of e n z y m e s y n t h e s i s after heating. Or, if r e p r e s s e r i n a c t i v a tion were r e a d i l y reversible, as t h e h e a t i n g e x p e r i m e n t m i g h t suggest, one w o u l d e x p e c t a n i m m e d i a t e r e c o v e r y a t 24 ° C a f t e r g r o w t h a t 42 ° C in t h e presence of n a h d i x i c acid. N e i t h e r is found. Moreover, t h e Q10 for t h e t r a n s i t i o n of i n d u c i b i l i t y

Thermosensitive D-Serine Deaminase Synthesis

377

to constitutivity is a b o u t 2.5. This is considerably lower t h a n the Q10 for the lac iTL m u t a n t (Sadler and Novick, 1965). We suggest t h a t the dsdC1 m u t a t i o n does specify a regulatory substance t h a t is heat-labile, as opposed to the p r o d u c t of its normal allele. This substance's low Q10, however, indicates t h a t a simple organic reaction, r a t h e r t h a n a protein denaturation, is involved in its functional inactivation. If the dsdCl p r o d u c t is a protein, this reaction might for example affect its interaction with another protein or with DNA. The possibility t h a t it involves formation of an endogenous inducer for D-serine deaminase seems excluded b y the fact t h a t the m u t a t i o n is cis dominant, trans recessive to its wild t y p e allele (McFall, 1967b). A gene affecting the production of an endogenous inducer would be expected to act b o t h cis and trans. W h a t e v e r its nature, the reaction m u s t be rather slow, to explain the lag in the a d j u s t m e n t of the rate of enzyme synthesis after the shift f r o m g r o w t h at 24 ° C to g r o w t h at 42 ° C. The lag which follows the shift from growth at 4 2 ° C to growth at 2 4 ° C could t h e n be due either to a slow reactivation of the dsdC1 product, or to the necessity for synthesis of new product. I t m a y be w o r t h noting t h a t the properties of this m u t a n t provide good evidence t h a t the D-serine deaminase system is under negative control. The fact t h a t dsdCl strains are inducible at low t e m p e r a t u r e and constitutive at high temperature is not consistent with positive control (Irr and Engelsberg, 1967). Acknowledgements. We are grateful to Drs. W. K. Maas and A. Novick for helpful discussions. This work was supported by U.S. Public Health Service research grant GM 11899. E. M. is a l~esearch Career Development Awardee U.S.P.H.S. GM 07390.

References Barbour, S., Gross, C, Noviek, A.: Growth instability of repressor. J. molec. Biol. 33, 967--969 (1968). Gallant, J., Stapleton, R. : Properties of a temperature sensitive regulatory system. Proc. nat. Acad. Sei. (Wash.) 50, 348--355 (1963). Goss, W., Dietz, W., Cook, T.: Mechanism of action of nalidixie acid on Escherichiacoli. J. Bact. 89, 1068--1074 (1965). Irr, F., Englesberg, E.: Revertants of nonsense mutants of the regulatory gene in the L-arabinose system of Escherichia coli B/r. Bact. Proe. 67, 54 (1967). MeFall, E. : Pleiotropic mutations in the D-serine deaminase system of Escherichia coll. J. molec. Biol. 9, 754--762 (1964). - - Mapping of the D-serine deaminase region in Escherichia coli K12. Genetics 55, 91--99 (1967a). - - Dominance studies with stable merodiploids in the D-serine deaminase system of Escherichia coli K12. J. Bact. 94, 1982--1988 (1967b). Roodyn, D. B., Mandel, It. G. : A simple membrane fractionation method for determining the distribution of radioactivity in chemical fractions of Bacillus subtilis. Biochim. biophys. Aeta (Amst.) 41, 80--88 (1960). Sadler, J. R., Novick, A. : The properties of repressor and the kinetics of its action. J. molec. Biol. 12, 305--327 (1965). Communicated b y W. Maas Prof. Elizabeth McFall Department of Microbiology New York University School of Medicine 550 First Avenue New York NY 10016/U.S.A. 26 Molec.Gem Genetics106