Ribosomal and soluble protein synthesis during a nutritional shift-up. Influence of cyclic AMP on...

322 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 97464

RIBOSOMAL AND SOLUBLE P R O T E I N SYNTHESIS DURING A NUTRI-

TIONAL S H I F T - U P

INFLUENCE OF CYCLIC AMP ON fl-GALACTOSIDASE ACTIVITY"

GRAHAM C A R P E N T E R AND BRUCE H. SELLS*"

Department of Biochemistry, St. Jude Children's Research Hospital and the University of Tennessee, Memphis, Tenn. 38Ioi (U.S.A.)

(Received May 3oth, 1972) (Revised manusc r ip t received August 25th, 1972)

SUMMARY

During the first 2o rain of a nutritional shift-up in Escherichia coli the ratio of ribosomal protein to soluble protein synthesis is increased to a level four times that characteristic of balanced growth. To determine whether this exaggerated synthesis of ribosomal protein imposed any restrictions on the formation of soluble protein the induced synthesis of fl-galactosidase was measured. Continuous synthesis of the enzyme was observed in the presence of io mM cyclic AMP which overcame the catabolite repression encountered in the shift-up medium. Studies on the influence of cyclic AMP upon the synthesis of ribosomal and total soluble protein revealed the cyclic nucleotides had no effect upon ribosomal protein synthesis while producing a marginal increase in the soluble protein formation. These investigations suggest that the exaggerated production of ribosomal protein during a shift-up does not restrict the synthesis of soluble protein.

Induction of fl-galactosidase during a shift-up at suboptimal concentrations of cyclic AMP (5 mM) resulted in a discontinuous pat tern of enzyme synthesis in a wild-type strain of E. coll. Studies with a phosphodiesterase-less mutant indicated that in the presence of 5 mM cyclic AMP thc synthesis of fl-galactosidase was continuous.

The addition of theophylline, an inhibitor of phosphodiesterase, to cells posses- sing an active phosphodiesterase increased the yield of fl-galactosidase at 5 mM cyclic AMP. These studies indicate that during a nutritional shift-up, when transient catabolite repression is effective, the activity of phosphodiesterase is implicated in regu- lating the synthesis of fl-galactosidase.

INTRODUCTION

Previous studies have established that the ribosome concentration in bacterial cells is proportional to the growth rate 1' ~ and that the rate of polypeptide elongation is independent of the growth rate 2'a. Thus, the level of general protein synthesis necessary to support a particular growth rate seems to be controlled by the number of

* A prel iminary repor t of this work was presented at the i l t h Annu. Meet. Am. Soc. Cell Biol., New Orleans, La., November 1971.

~ To whom enquiries should be addressed; at the Laborator ies of Molecular Biology, Faculty of Medicine, Memorial University, St. John ' s , Newfoundland, Canada.

Biochim. Biophys. Acta, 287 (1972) 322-329

CYCLIC AMP AND ~-GALACTOSIDASE ACTIVITY 323

ribosomes in the cell. The immediate response of bacterial cells to a nutritional shift- up is the production of additional ribosomal RNA and ribosomal protein 4-7. Although the preferential formation of ribosomal protein over soluble protein during a transition to a new growth rate has been observed by numerous investigators, little is known about mechanisms involved in controlling this preferential synthesis. The present experiments were designed to examine the ability of cells to synthesize fl-galactosidase, a soluble protein, during a shift-up and to determine whether the increased rate of ribosomal protein synthesis imposed restrictions on the synthesis of soluble proteins.

MATERIALS AND METHODS

Escherichia coli HI28 (leu-, arg-, thr- , his-, met-, thi-, rel÷) and E. coli B were obtained from Dr Herbert L. Ennis; E. coli strain AB257 (met-) and AB257pc -1 (met-, cyclic AMP phosphodiesterase negative) were obtained from H. V. Rickenberg. This latter mutant possesses only 5 % of the phosphodiesterase activity of the wild type. Cultures of HI28 and AB257 were grown with shaking at 30 °C in either Cohen's 8 or M99 minimal medium, respectively. The preshift medium contained 0. 4 °/o potas- sium acetate and required supplements. Enriched medium contained 0.4 % glucose, all amino acids (50/zg/ml) and nucleosides (20 #g/m1). Transitions between media were carried out by centrifuging cells from the preshift medium, resuspending in cold minimal medium, warming quickly to 3 ° °C, and adding an appropriate aliquot to prewarmed enriched medium. This procedure took about 15 min. Alternatively, enrichments were added directly to the minimal medium. No difference was observed between the two procedures. Cell growth was measured by the increase in absorbance at 575 nm. The assay of fl-galactosidase activity was carried out as described by Pardee et al. ~°. Isopropylthiogalactoside was used at a concentration of 5 " lO-4 M. The specific activity of ribosomal and soluble protein fractions were determined as described by Davis and Sells ~1. E14ClLysine, specific activity 31o Ci/mole, and cyclic 3',5'-adenosine monophosphate were obtained from Schwarz BioResearch Co. Iso- propylthiogalactoside was a product of Calbiochem.

RESULTS

When cells growing in the acetate medium are transferred to the enriched medium the synthesis of ribosomal protein is preferentially increased. This observa- tion is illustrated in Fig. i. In this experiment the incorporation of [14C]lysine into ribosomal and soluble protein was determined at intervals following the shift-up. Since a lag occurs between the time ribosomal proteins are synthesized and the time they appear on mature particles, each I-min labeling period was followed by a 3o-min incubation with an excess of unlabeled lysine to insure complete assemblage of the ribosomal proteins into the ribosomes. These studies demonstrate that 20 rain following the shitt-up the ratio of ribosomal to soluble protein synthesis had reached a value four times that characteristic of balanced growth. Similar pulse-chase experiments performed to determine the rate of synthesis of soluble and ribosomal protein during balanced growth in acetate medium and in an enriched medium revealed that

Biochim. Biophys. Acta, 287 (I972) 322-329

324 G. CARPENTER, B. H. SELLS

5o[ 6 0 0

5OO

._c

2 4 0 0

<

n_ 3 0 0

2 0 0

100

(524)

• 2 )

Lo8)

0 4_ ~ l _ i i J 0 I0 20 30 4 0 50 6 0

MINUTES A~ TER SHIFT UP

Fig. I. Syn the s i s of r ibosomal and soluble p ro te in du r ing a sh i f t -up in E. coli H128. ( 0 ) Ribo- somal prote in; (O) soluble protein. N u m b e r s in pa r en t he se s indicate ra t io of r ibosomal to soluble p ro te in syn thes i s .

although there was a 3-fold difference in the generation times of the cells in the two media there was no difference in the ratios of ribosomal to soluble protein synthesis (see Table I). These investigations indicate that (I) during balanced growth the relative rate of synthesis of ribosomal to soluble protein is independent of growth rate and that (2) during an upward shift in growth rate a rapid increase in ribosomal protein synthesis occurs prior to an increase in soluble protein synthesis.

T A B L E I

G R O W T H C H A R A C T E R I S T I C S D U R I N G B A L A N C E D G R O W T H

Cul tu res of E. coli H I 2 8 were m a i n t a i n e d in exponen t i a l g rowth in ace ta te and enr iched media . E a c h cu l tu re was pu lsed for i m i n w i t h L-[14CJlysine (o.i mCi /ml and 7 " l ° -8 g/ml). The label was t h e n chased by add ing non- rad ioac t ive lys ine (15oo ffg/ml). The cu l tu res were i ncuba t ed for 3 ° m i n and t h e n poured over c rushed ice, cen t r i fuged and specific ac t iv i t ies of t he r ibosomal and soluble p ro te in f rac t ions de te rmined .

Acetate media Enriched media

Genera t i on t ime (min) 165 R ibosoma l p ro t e in s y n t h e s i s (cpm//zg) 298 Soluble p ro te in s y n t h e s i s (cpm//zg) 231 R ibosoma l p ro te in syn t he s i s 1.28 Soluble p ro te in s y n t h e s i s

6 o

54 ° 43 °

1.25

fl-Galaetosidase synthesis during shi]t-up To determine whether the increased synthesis of ribosomal protein during

shift-up imposed restrictions on the synthesis of soluble protein the ability of cells to synthesize fl-galactosidase was examined. Initial experiments revealed that enzyme induction occurred only when cyclic AMP was added to overcome the catabolite

Biochim. Biophys . Acta, 287 (I972) 322-329

CYCLIC AMP AND /~-GALACTOSIDASE ACTIVITY 325

repression encountered in the glucose medium. Consequently a suspension of E. coli HI28 growing exponentially in pre-shift medium was shifted into a flask containing enriched medium, isopropylthiogalactoside and IO mM cyclic AMP. Under these conditions, induction and continuous enzyme synthesis were observed (see Fig. 2). This s tudy suggests that the exaggerated rate of synthesis of ribosomal protein does not limit the synthesis of soluble protein.

4 0 0

- /

300 10 mM CYCLIC A M P ~

2 v- o 5ram

200 o o

1ram CYCLIC AMP I00 A ~ A

0 0 3 0 6 0

MINUTES AFTER SHIFT-UP

Fig. 2. Induction of /~-galactosidase during a shift-up in E. coli 1-1128, Io mM cyclic AMP ( t ) 5 mM cyclic AMP (0), I mM cyclic AMP (LX), minus cyclic AMP (&).

Influence o/cyclic A M P upon synthesis o] ribosomal and soluble protein Since cyclic AMP is necessary for the synthesis of ~-galactosidase, the following

experiments were performed to determine whether the presence of cyclic nucleotide produced a change in the overall rate of soluble or ribosomal protein synthesis. A culture of E. coli HI28 growing exponentially in preshift medium was shifted into four flasks containing enriched media. Two of the flasks were supplemented with cyclic AMP. At i0 and 40 min after the shift-up two cultures, with and without cyclic AMP, received L-[14C]lysine. 1 min later an excess of unlabeled lysine was added and the incubation continued for an additional 3 ° rain. The cells were poured over crushed ice, centrifuged, processed and the specific activities of the ribosomal and soluble protein fractions determined. The data presented in Table I I indicate that the presence of cyclic AMP had no influence upon the rate of synthesis of ribosomal protein and produced a marginal increase (10-20 %) in soluble protein synthesis. These results suggest that the exaggerated production of ribosomal protein during shift-up is not influenced by the presence of cyclic AMP even though soluble protein synthesis is slightly stimulated.

Synthesis o~ fl-galactosidase during shift-up in the presence o~ limiting concentration o/cyclic A M P

To determine the effect of limiting concentrations of cyclic AMP upon fl-galac-

Biochim. Biophys. Acha, 287 (1972) 322-329

3 2 6 G. CARPENTER, B. H. SELLS

T A B L E I I

EFFECT OF IO mM CYCLIC AMP UPON THE SYNTHESIS OF RIBOSOMAL AND SOLUBLE PROTEIN DUR- ING A SHIFT-UP

A cu l tu re of E. coli growing e x p o n e n t i a l l y in p resh i f t m e d i u m was sh i f ted in to four f lasks con- t a i n i n g enr iched media . Two of the f lasks were s u p p l e m e n t e d w i t h cyclic AMP ( io raM). At i o and 4 ° min a f te r the sh i f t -up two cul tures , plus and minus cyclic AMP, rece ived L-E14Cllysine (o.i #Ci /ml and 7 " IO-S g/ml) . I rain l a t e r the label was chased by a d d i n g un labe led lys ine (15oo /~g/ml) and i n c u b a t i o n con t inued for an a d d i t i o n a l 3 ° rain. The cu l tu res then were poured over c rushed ice, cen t r i fuged and the specific ac t iv i t i e s of the r ibosomal and soluble p ro te in f rac t ions de te rmined .

Min after Added cyclic A M P Spec. act. (cpm/mg) shift-up (raM)

Ribosomal protein Soluble protein

IO IO 497 169 IO None 502 139

40 IO 55o i63 4 ° None 54 ° 147

tosidase during a shift-up cells growing exponentially in preshift medium were shifted into three flasks containing enriched medium, isopropylthiogalactoside and varying concentrations of cyclic AMP. In the presence of io mM cyclic AMP induction and continuous synthesis occurred; however, in the presence of i mM cyclic AMP only limited enzyme synthesis occurred during the first 20 min (see Fig. 2) after which time fl-galactosidase formation was arrested.

With an intermediate concentration (5 raM) of cyclic AMP a discontinuous pattern of enzyme synthesis was observed. Enzyme formation was continuous for the first 25 min, arrested for 30 rain and then continuous. Fig. 3 illustrates this discontinuous synthesis over a longer time period in the presence of 3 mM cyclic AMP. This discontinuous pattern does not occur if fl-galactosidase is induced during balanced growth (see Fig. 4).

Phosphodiesterase activity and the discoutinuous pattern o[ fl-galactosidase synthesis A number of investigators have concluded that fl-galactosidase synthesis is

controlled in part by the intracellular level of cyclic AMP 12-14. Relatively little is known, however, concerning the regulation of intracellular levels of cyclic AMP. One point of regulation involves the activity of the enzyme phosphodiesterase which hydrolyzes cyclic AMP to 5'-AMP + PI . TO determine whether a modification of phosphodiesterase activity might account for the discontinuous pattern of fl-galactosidase synthesis, induction during the shift-up was performed in the presence of 5 mM cyclic AMP and theophylline, an inhibitor of phosphodiesterase. The results shown in Fig. 5 indicate that in the presence of 2 mM theophylline the pattern of fl-galactosidase synthesis is modified. While the previously described pattern of discontinuous enzyme synthesis is observed in the control culture, theophyUine extends the initial period of continuous enzyme synthesis until 60 rain after the shift-up and increases the amount of enzyme synthesized. At a concentration of 2 mM theophylline did not effect the growth rate although higher concentrations were toxic. Theophylline apparently increases the intracellular level of cyclic AMP as iudged by its effect upon fl-galactosidase synthesis.


CYCLIC AMP AND ~-GALACTOSIDASE ACTIVITY

50O

327

5 0 0

u~

2 0 0

>-

uJ

4 0 0 o

o o

o

IO0

O ~ [ I I I l l _ _ L L l I 1 ~ L ! . ~ 0 30 60 9 O

MINUTES AFTER SHiFT-UP

I 0 0 0

I - -

D 7 5 0 -<

== uJ 5 0 0

=- uJ

250 • • o

3 0 6 0 MINUTES AFTER INDUCTION

Fig. 3. Induction of /5-galactosidase in the presence of 3 mM cyclic AMP during the first 2 h of a shift upon E. coli HI28.

Fig. 4- Induction of ~-glactosidase during balanced growth of E. coli HI28 in enriched media, io mM cyclic AMP(&), 5 mM cyclic AMP (A), i mM cyclic AMP (O), minus cyclic AMP (@).

4 0 0

~ 3 0 0 ~

o

200 ~ o

g

>-

~ 1 0 0

0 30 60 9O MINUTES A~'TER SHIFT-UP

Fig. 5. Effect of theophylline on the induction of/5-galactosidase by E. coli HI28 in the presence of 5 mM cyclic AMP. Minus theophylline ( 0 ) , 2 mM theophylline (A).

Since complete inhibition of phosphodiesterase cannot be achieved with them phylline 1~ experiments were performed with a mutant lacking phosphodiesterase activity. The influence of cyclic AMP concentration on the induction of /5-galactosidase in AB257pc -1 a phosphodiesterase mutant is shown in Figs 6 and 7, respectively. In the presence of 4 mM cyclic AMP, continuous enzyme synthesis is observed in the diesterase-negative mutant (Fig. 7) while discontinuous synthesis is observed in the parent (Fig. 6). Results similar to those obtained with AB257pc -1 have been obtained using E. coli B which also lacks phosphodiesterase activity 15.

Bio6him. Biophys. Acta, 287 (1972) 322-329

328 G. CARPENTER, B. H. SELLS

~000 I

800 l

z

6OO

L~ 400

t~

2OO

O-

600

500

L :,o ~' ~ 4 0 0

~ 300

t~ 200 o o

IO0

3 0 6 0 9 0 120

MINUTES AFTER SHIFT-UP

z~

30 60 90 120 MINUTES AFTER SHIFT-UP

Fig. 6. I n d u c t i o n of f l -galactosidase in AB257 du r i ng a sh i f t -up . 4 mM cyclic AMP (Q) , I m M cyclic A M P (O) , o.I m M cyclic A M P (,~).

F ig • 7. I n d u c t i o n of f l -galactosidase in AB257pc -x du r ing a sh i f t -up . 4 m M cyclic AMP ( 0 ) , I m M cyclic A M P (O) , o. i m M cyclic A M P (A) .

At lower concentrations, I mM and o.I mM of cyclic AMP, the parent strain AB257 which possesses phosphodiesterase activity forms very little fl-galactosidase during the first 30 rain of shift-up. In the mutant the rate of synthesis of fl-galactosidase during the first 3 ° rain is similar at all concentrations of the cyclic nucleotide employed. Thus, at low concentrations of cyclic AMP the influence of phosphodiesterase becomes evident in the parent strain and apparently acts to reduce the intracellular concentration of cyclic AMP and thereby restricting the synthesis' of fl- galactosidase. In the mutant, however, fractionating the concentration has no effect upon the initial rate of fl-galactosidase synthesis. The effect of the different levels of cyclic AMP becomes evident in the mutant only after the first 30 rain when the levels of fl-galaetosidase synthesis are a reflection of the concentrations of cyclic AMP in the medium.

These later observations might indicate that in the mutant, lowering of intracellular cyclic AMP levels may be affected by a transport mechanism while in the parent strain both transport and phosphodiesterase are involved. Furthermore, during shift-up it takes approximately 30 rain to lower the intracellular concentration of cyclic AMP below the threshold level required for fl-galactosidase synthesis in the mutant.

Studies were undertaken to examine phosphodiesterase activity of wild-type cells obtained from cultures exhibiting discontinuous synthesis of fl-galactosidase. The results, however, indicated little significant change in enzyme level in broken cell extracts. From these data, therefore, it is not possible to conclude that the activity of phosphodiesterase fluctuates during the discontinuous synthesis of fl-galactosidase. These results do not eliminate the possibility, however, that there are intracellular


CYCLIC A M P AND fl-GALACTOSIDASE ACTIVITY 329

changes in the a c t i v i t y of phosphodies te rase which cannot be de tec ted when the cells are d i s rupted .

DISCUSSION

E x a m i n a t i o n of the induct ion of f l -galactosidase dur ing the ear ly per iod of the shi f t -up process indica tes t h a t the preferent ia l synthes is of r ibosomal pro te in does not t ake place a t the expense of factors requi red for soluble pro te in synthesis . Follo- wing t ransfer of cells from a poor to an enr iched med ium ca tabo l i t e repression was observed, and synthes is of f l -galactosidase a t a cont inuous ra te could only be achieved b y the add i t i on of cyclic A M P at high concent ra t ions (I0 raM). A t a subop t ima l concen t ra t ion (5 mM) a d iscont inuous p a t t e r n of enzyme synthes is was observed.

Inves t iga t ions of the d iscont inuous synthes is of f l -galactosidase observed at s ubop t ima l cyclic AMP levels i nd i ca t ed t ha t phosphodies te rase m a y be impl i ca ted in these results . Suppor t for th is a rgument was ob ta ined ( i ) b y the use of the inh ib i tor of phosphodies te rase , theophyl l ine , and (2) b y the use of a m u t a n t E. coli lacking phosphodies terase . If the phosphodies te rase level is reduced or i ts a c t i v i t y pa r t i a l l y inh ib i t ed dur ing the shif t -up, the y ie ld of f l -galactosidase is increased. These observa t ions suggest t h a t the a c t i v i t y of phosphodies te rase m a y f luc tua te under the shi f t -up condi t ions. F luc tua t i ons in the a c t i v i t y of phosphodies te rase would resul t in a modu- la t ion of the in t race l lu la r concent ra t ion of cyclic AMP.

ACKNOWLEDGEMENTS

These s tudies were suppo r t ed b y the U. S. Publ ic Hea l t h Service Gran t AM- 07375, and the D a m o n R u n y o n Memorial Fund . G. C. was a research t ra inee on the Na t iona l Cancer I n s t i t u t e Tra in ing Gran t ToI-CA-05176. The technica l ass is tance of Mrs D iana Jones is g ra te fu l ly acknowledged. We t h a n k Dr George Cheung for the measu remen t of phosphodies te rase a c t i v i t y in broken cell ext rac ts .

REFERENCES

I R. E. Ecker and M. Schaechter, Biochim. Biophys. Acta, 76 (1963) 275. 2 R. Rosset, J. Julien and R. Monier, J. Mol. Biol., 18 (1966) 308. 3 J. Forchhammer and L. Lindahl, J. Mol. Biol., 55 (1971) 563 • 4 N. O. Kjeldgaard, O. Maaloe and M. Schaechter, J. Gen. Microbiol., 19 (1958) 607. 5 N, O. Kjeldgaard, Biochim. Biophys. Acta, 49 (1961) 64. 6 R. Schlief, J. Mol. Biol., 27 (1967) 41. 7 R. J. Harvey, J. Bacteriol., IOI (197o) 574. 8 S. S. Cohen and R. Arbogast, J. Exp. Med., 91 (195o) 619. 9 M. Inouye and A. B. Pardee, J. Bacteriol., io i (197 o) 77 o.

IO A. B. Pardee, F. Jacob and J. Monod, J. Mol. Biol., i (1959) 165. I i F. C. Davis and B. H. Sells, J. Mol. Biol., 39 (1969) 5o3 • 12 R. Perlman and I. Pastan, Biochem. Biophys. Res. Commun., 3o (1968) 656. 13 A. Ullmann and J. Monod, FEBS Lett., 2 (1968) 57. 14 M. Aboud and M. Burger, Biochem. Biophys. Res. Commun., 43 (197 o) 174. 15 I. Pastan and R. Perlman, Science, 169 (197o) 339.


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