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Requirements for K+ for in vivo synthesis of Escherichia coli and Bacillus subtilis cell constituents
Bioehimica et Biophysica Acta, 294 (1973) 87-93 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
87
BBA 97492
R E Q U I R E M E N T S FOR K + FOR I N V I V O SYNTHESIS OF ESCHERICHIA
C O L I AND B A C I L L U S
SUBTILIS
CELL CONSTITUENTS
HERBERT L. ENNIS, KATHARINE D. KIEVITT AND ROBERT F. PETERSON" Roche Institute of Molecular Biology, Nutley, N.J. o7zzo (U.S.A.) (Received July 3rd, 1972) (Revised manuscript received September I ith, I972)
SUMMARY Mutants of Escherichia coli and Bacillus subtiIis which have lost the normal capacity to accumulate K + from the growth medium are useful for studying the i n vivo requirement for K + for the synthesis of cell components. The intracellular K+ concentration can be regulated very precisely ill these mutants by varying the concentration of the cation in the growth medium. When these mutants are depleted of their internal K + growth ceases. This investigation shows that K÷ depletion has a pleiotropic effect on the synthesis of a variety of cell components. Protein, total lipid, and acid-soluble nucleotide synthesis are inhibited in K+-depleted cells. The inhibition of lipid and nucleotide synthesis is not due to the cessation of protein formation, because their synthesis continues in chloramphenicol-treated cells. On the other hand, RNA, DNA and to a limited extent murein synthesis continues in the absence of K +. RNA synthesis in K+-depleted cells appears to be limited by the availability of purines and pyrimidines and consequently the addition of these compounds in the starvation medium markedly stimulates RNA formation.
INTRODUCTION Mutants of Escherichia coli B (ref. I) and KI2 (ref. 2) and Bacillus subtilis 3 which have lost the normal capacity to transport and concentrate K + from the growth medium have been isolated. The internal K + concentration can be regulated very precisely in these mutants by merely varying the concentration of the cation in the growth medium 4. It is a very simple and rapid procedure to deplete cells of their internal K+. These mutants are very useful for studying the in vivo requirement for K+ for the synthesis of cell components, because they are the only way one can readily regulate the intracellular K ÷ concentration. Microbial cells require K + for growth and protein synthesis 1,4,5. In contrast to protein synthesis, RNA synthesis continues in K÷-depleted cells 4,s-s. It is known that several E. coli enzymes require K + for activity 9. For example, several K +requiring enzymes are involved in the synthesis of pufines 1° and pyrimidines n. * Present address: Sloan-Kettering Institute for Cancer Research, New York, N.Y. (U.S.A.)
~N
It. L. I(NNI> ~'l d l .
Since we are able t(, easily regulate the intracellular 1,2 concentrati(m \re at,. in a position to study some .f these reactions in ~,i;,~, in an attempt to determine what K'-requiring reaction is growth limiting. The present study is a survey o1 the in ~'iv,() requirement for t,: for the synthesis of a variety of cell components.
MATERIALS AND METHOI)S
Bach,rial strains, media and growth conditions E. coli strain B2o 7 and B. subtilis 168 K\V mutants defective in their ability to concentrate and accunmlate K ¢ from the growth medium were used ]'3'12. The cells were grown in K ~-containing nmdium A la, supplemented with glucose (o.25 tl~,) and histidine, leucine and methiolfine (IOOktg/ml each) in the case of B2o7, and in the same medium supplemented with 0.2 ° o casamino acids and thymine and tryptophan (20/~g/'ml each) for 14. s~tbtilis. In medimn lacking K~, the potassium phosphates were replaced by an equinlolar concentration of sodium phosphates. All cultures were grown at 37 ~'C with vigorous aeration and were used at approximatel 3 5 "IOS cells per nil. Unless otherwise indicated all media contained adenine (or adenosine), guanine (or guanosine), cytidine and uracil at 20 #g/ml each. Growing cells were centrifuged at 3o00 x g in a Sorvall SP Table Top centrifuge at room temperature, and were washed free of K ~ by two successive resuspensions and sedimentations. The cells were then resuspended it] the appropriate medium indicated ill each experiment.
Determination o/ the synthesis o/ cell components The synthesis of RNA ~, protein 4, DNA 14, murein la, acid-soluble nucleotides t6 and lipid lr were determined as previously described. Growth was determined by increase in turbidity using a Klett-Summerson photoelectric colorimeter (No. 42 filter), ioo Klett units corresponds to 5 ' I°8 cells per ml.
Radioisotopes [2J4C]Uracil (50.3 Ci/mole), [8-1aC_]adenine (52 Ci/nlole), I.iU-14C]leucine (202 Ci/mole), DLU-14C]glucose (I5.5Ci/mole), L-~UJ4C]alanine (I28Ci/mole), and [2-14Cithymine (28 el/mole) were purchased from New England Nuclear Corp., Boston, Mass.
RESULTS
EHect o / K + depletion on growth and synthesis o/cell components The requirement for K+ for growth and the synthesis of a variety of cell components is presented in Fig. I. K+ is required for the growth of E. coli strain B2o 7 (Fig. IA). In the absence of K +, there is practically no increase in absorbance. Similarly, protein synthesis is almost completely inhibited in the absence of K + (Fig. IB). These two findings have been well documented previously 1'~ and are presented here only for heuristic purposes. The synthesis of acid-soluble nucleotides is severely inhibited in cells depleted of K + (Fig. IC). That this is not a function of the lack of protein synthesis in the absence of K + is shown by the observation that
K + REQUIREMENT FOR in vivo SYNTHESIS 3OO
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120
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DNA
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15 30 4.5 60 MINUTES AFTER ADDITION OF [14C~THYMINE
O
,
~
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15 30 45 60 MINUTES AFTER ADDITION OF [14C']ALANINE
Fig. i. Requirement for K+ for growth and synthesis of cell components. Exponentially growing cells were depleted of K+ as described in Materials and Methods. All methods for measurements and determinations are described in Materials and Methods. Where indicated chloramphenicol (CM) was used at IOO/~g/ml. Incorporation of [*4C]glucose into acid-soluble nucleotides was determined as previously described*t The cells were incubated under the indicated conditions in the presence of 2 mM [z~C]glucose. At the indicated times i-ml aliquots were added to 2 ml cold 7.5 % perchloric acid. After 60 rain standing in the cold the precipitate was collected by centrifugation and was washed once with 2 ml cold 5 % perchloric acid. The supernatant and wash were combined and o.2 ml 15 ~o Norit was added. The Norit was collected by filtration through Millipore filters (0.45 #m pore size), washed with cold 5 % perchloric acid, dried, and counted in a Beckman Liquid Scintillation spectrometer in Omnifluor. the rate of acid-soluble nucleotide synthesis b y cells i n c u b a t e d in the presence of K ÷ a n d chloramphenicol is initially almost n o r m a l (Fig. IC). At this c o n c e n t r a t i o n chloramphenicol almost completely inhibits protein synthesis a n d growth. T o t a l lipid synthesis is severely depressed in cells i n c u b a t e d in the absence of K + (Fig. ID). These results were also o b t a i n e d using incorporation of ~ P as an i n d e p e n d e n t measure of lipid synthesis. Lipid synthesis continues in cells i n c u b a t e d with c h l o r a m phenicol, although at a somewhat diminished rate compared to growing cells (Fig ID). D N A synthesis (Fig. I E ) in the absence of K + proceeds at a b o u t one-half the rate observed in e x p o n e n t i a l l y - g r o w i n g cells (see also refs. 8 a n d 14). Murein synthesis continues b u t at a slower rate t h a n n o r m a l in K+-depleted cells for a b o u t 30 m i n a n d t h e n a b r u p t l y stops (Fig. I F ) .
{)0
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I)ltriJzcs a~zd pyrimidim:s stimztlah~ the s_wzthcsis o~ RA'A in l£ -dc/)k,&d cc/l,s RNA synthesis continues in cells depleted of K (see refs 4 and (~ 8). How plete amino acid mixture or thymine to K ~-depleted cells did not stimulate u C ] uracil incorporation over that amount observed in the absence ~d! purinc. 70
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.
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[14C]URACIL
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15 30 45 60 MINUTES AFTER ADDITION OF [14C]ADENINE
Fig. 2. Stimulation b y purines a n d pyrimidines of R N A synthesis in E. coil in the absence of K+. (A) Growing cells of strain B2o 7 were depleted of K~ as described in Materials and Methods. [t4C]Uracil (o. 3 ~Ci/mI; io/~g/ml) was added and samples were t a k e n at the indicated intervals. I n c o r p o r a t i o n of [14C]uracil into R N A was carried o u t as described in Materials and Methods. A growing culture was similarly handled. 2o Mg/ml non-radioactive adenine or guanine were used where indicated. (B) This e x p e r i m e n t was the same as outlined in (A) except t h a t [14C]adenine (o. 3/~Ci/ml; i o / z g / m l ) and 2o/*g/ml non-radioactive uracil were used. K+-depleted cells plus uracil (@) ; K+-depleted cells, no uracil ( • ) .
91
K + REQUIREMENT FOR in vivo SYNTHESIS
That uracil is also required for maximum stimulation of RNA synthesis in K+-depleted cells is shown in Fig. 2B. In this experiment the incorporation of [z4C] adenine into RNA was determined in the presence and absence of non-radioactive uracil. As can be seen, the presence of uracil greatly stimulated the incorporation of [l*C]adenine. As seen in Fig. 3, purines and pyrimidines are also required for maximum stimulation of RNA synthesis during K+ depletion of a B. subtilis mutant defective in K ÷ accumulation. In this mutant, adenosine (or adenine or inosine) alone stimulates the incorporation of [14C]uracil much better than guanosine (or guanine) alone. The addition of both adenosine and guanosine gave no more stimulation than observed using adenosine alone (data not shown). We previously showed that RNA synthesis proceeded during bacteriophage T 4 infection of E. coli B2o 7 in the absence of K ÷ (see ref. 18). The data presented in Fig. 4 show that added purines and pyrimidines stimulate the synthesis of RNA in cells infected in the absence of K ÷ over 3-fold that observed in the absence of compounds. No significant stimulation by added purines and pyrimidines of RNA synthesis during infection in the presence of K ÷ was noted. 75
150
RNA
/
RNA E_.COLI /
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~
'---" " 45
90
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30
45
60
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MINUTES AFTER ADDITION
MINUTES AFTER ADDITION
OF [14C]URACIL
OF [14C] URACIL
Fig. 3. Stimulation b y p u r i n e s a n d pyrimidines of R N A synthesis in B. subtilis in t h e absence of K +. This e x p e r i m e n t was p e r f o r m e d as outlined in Fig. 2A, u s i n g B. subtilis s t r a i n K W . Fig. 4. S t i m u l a t i o n b y p u r i n e s and pyrimidines of R N A synthesis in E. coli infected w i t h bacteriophage T 4 in the absence of K+. Growing cells or K+-depleted cells of s t r a i n B2o 7 were infected w i t h bacteriophage T 4 a t a multiplicity of 5 bacteriophage per cell as previously described 31. Less t h a n i % of the cells infected survived. I n c o r p o r a t i o n of [14C]uracil into R N A (exclusive of bacteriophage DNA) was determined as previously described St.
DISCUSSION
By use of mutants of E. coli and B. subtilis defective in their ability to accumulate K+ from the growth medium, we have been able to study the in vivo requirement
02
H. 1.. I. NNIS ,'/ ~g/.
for this cation for the synthesis of a variety of cells components. K depletion is a simple and rapid procedure. Cells are viable for long periods of time in the ab>ence of K " (see ret. 8). ]'lie unique feature of this m e t h o d (as shown by previous w,,rk a) is t h a t one can maintain a very fine control over the intracellular K ~ concentration merely by the simple manipulation of the anlount of K I in the growth nledium. To our knowledge this is the only method available for this purpose. One need only add back K - to the depleted cell culture to reiniate growth. Since it is kn,~wn that several enzymes require K~ for activity or are stimulated by the cation we were in a position to s t u d y these reactions, anti others, i~, vivo. We had previously shown t h a t K ¢ was required for growth and protein swlthesis *'4. In the present investigation we further delnonstrate that K is also required for the synthesis of purines and pyrinfidines. This confirms work done on the K requirement for the i,a vitro activity of certain enzyines involved in purine and pyrinfidine synthesis ~°'n. When purines and pyrimidines are added to cultures of cells depleted of K +, R N A synthesis is stimulated by approximately 4-6 fold t h a t observed in depleted cells lacking the bases. Consequently, although K ~ has been implicated in tim M vitro formation of G T P and d G T P fronl G D P and d G D P respectively l'q, it is obvious t h a t enough GTP is made i,z vivo, perhaps by an alternative lmthway, to supply some of the G T P and d G T P needed for RNA and D N A synthesis, resl)ectively. D N A synthesis in the absence of K+ proceeds at about one-half the initial rate compared to growing cells, (see also ref. i4). Murein synthesis also proceeds in the absence of K+ but only to a limited extent. More importantly, total lipid synthesis is ahnost completely inhibited in the absence of K +. The consequences of this defect on cell metabolisin is not known. This observation is in agreement with previous work which showed t h a t K r (but not Na +) was needed for CDP-diglyeeride formation in microorganisms '0 "2. Perhaps K~ is also required for an earlier step in lipid synthesis. K - has been reported to be necessary for the in vitro activity of ribonuclease I I 2a "~" of E. coll. This enzyme m a y be involved in messenger R N A decay in vivo as well as i~a vitro and consequently m a y have important regulatory significance "a,27 a0. This can be studied by use of the K+-dependent n m t a n t s with the aim of stabilizing messenger R N A under conditions of K + depletion. Since we have already shown that K + is required for bacteriophage T 4 messenger R N A breakdown is, we are now studying the role of K + in E. coli messenger R N A nletabolism.
REFERENCES M. L u b i n a n d D. Kessel, t3iochem. Biophys. Res. Commun., 2 (196o) 249. W. E p s t e i n a n d B. S. Kim, .[. Bactcriol., i o 8 (1971) 639. M. L u b i n , Fed. Proc., 23 (1964) 994. M. L u b i n a n d H. L. Ennis, Biochim. Biophys. dcta, 80 (1964) 614. G. L ester, J. BacterioL, 75 (1958) 426. H. L. E n n i s a n d M. L u b i n , Biochim. Biophys. Acta, 95 (1965) 605. H. L. E n n i s a n d M. L u b i n , Biochim. Biophys. Acta, 95 (I965) 624. H. L. E n n i s a n d M. L u b i n , Biochim. Biophys. Acta, 5o (1961) 399. C. H. Seulter, Science, 168 (197 o) 789. J. M. B u c h a n a n , 7"he Harvey Lectures, 1958-1959, Series LIV , Acadenfic Press, N e w Y o r k , I96O, p. lO4. I I P. M. A n d e r s o n a n d A. Meister, Biochemistry, 5 (196o) 3157 . I 2 3 4 5 6 7 8 9 io
K + REQUIREMENT FOR in viuo SYNTHESIS 12 13 14 15 16 17 18 19 2o 21 22 23 24 25 26 27 28 29 3°
93
D. B. Willis and H. L. Ennis, J. Virol, 3 (1969) i. B. D. Davis and E. S. Mingioli, J. Bacteriol,, 6o (195 o) 17. K. E. Rubenstein, E. Streibel, S. Massey, L. Lapi and S. S. Cohen, J. Bacteriol., in the press. E. J. J. Lugtenberg and P. G. De Haan, Antonie van Leeuwenhoek, J. Microbiol. Serol., 37 (1971) 537. Y. Sokawa, E. Nakao-Sato and Y. Kaziro, Biochim. Biophys. Acta, 199 (197 o) 256. J. Kanfer and E. P. Kennedy, J. Biol. Chem., 239 (1964) 172o. P. S. Cohen and H. L. Ennis, Virology, 27 (1965) 282. M. P. Oeschger and M. J. Bessman, J. Biol. Chem., 241 (1966) 5452. J. t{. Carter, J. Lipid Res., 9 (1968) 748. R. E. McCaman and W. R. Finnerty, J. Biol. Chem., 243 (1968) 5o74. P. H. Patterson and W. J. Lennarz, J. Biol. Chem., 246 (1971) lO62. P. F. Spahr, J . Biol. Chem., 239 (1964) 3716. P. F. Spahr and D. Schlessinger, J. Biol. Chem., 238 (1963) PC225I. M. F. Singer and G. Tolbert, Science, 145 (1969) 593M. F. Singer and G. Tolbert, Biochemistry, 4 (1965) 1319. T. Kivity-Vogel and D. Elson, Biochem. Biophys. Res. Commun., 33 (1968) 412. M. Artman, N. Silman and H. Engelberg, Biochem. J., lO 4 (1967) 878. H. O. Voorma and L. Bosch, Biochem. Biophys. Res. Commun., 18 (1965) 42o. E. T. Lennette, L. Gorelic and D. Apirion, Proc. Natl. Acad. Sci. U.S., 68 (1971) 314o..
87
BBA 97492
R E Q U I R E M E N T S FOR K + FOR I N V I V O SYNTHESIS OF ESCHERICHIA
C O L I AND B A C I L L U S
SUBTILIS
CELL CONSTITUENTS
HERBERT L. ENNIS, KATHARINE D. KIEVITT AND ROBERT F. PETERSON" Roche Institute of Molecular Biology, Nutley, N.J. o7zzo (U.S.A.) (Received July 3rd, 1972) (Revised manuscript received September I ith, I972)
SUMMARY Mutants of Escherichia coli and Bacillus subtiIis which have lost the normal capacity to accumulate K + from the growth medium are useful for studying the i n vivo requirement for K + for the synthesis of cell components. The intracellular K+ concentration can be regulated very precisely ill these mutants by varying the concentration of the cation in the growth medium. When these mutants are depleted of their internal K + growth ceases. This investigation shows that K÷ depletion has a pleiotropic effect on the synthesis of a variety of cell components. Protein, total lipid, and acid-soluble nucleotide synthesis are inhibited in K+-depleted cells. The inhibition of lipid and nucleotide synthesis is not due to the cessation of protein formation, because their synthesis continues in chloramphenicol-treated cells. On the other hand, RNA, DNA and to a limited extent murein synthesis continues in the absence of K +. RNA synthesis in K+-depleted cells appears to be limited by the availability of purines and pyrimidines and consequently the addition of these compounds in the starvation medium markedly stimulates RNA formation.
INTRODUCTION Mutants of Escherichia coli B (ref. I) and KI2 (ref. 2) and Bacillus subtilis 3 which have lost the normal capacity to transport and concentrate K + from the growth medium have been isolated. The internal K + concentration can be regulated very precisely in these mutants by merely varying the concentration of the cation in the growth medium 4. It is a very simple and rapid procedure to deplete cells of their internal K+. These mutants are very useful for studying the in vivo requirement for K+ for the synthesis of cell components, because they are the only way one can readily regulate the intracellular K ÷ concentration. Microbial cells require K + for growth and protein synthesis 1,4,5. In contrast to protein synthesis, RNA synthesis continues in K÷-depleted cells 4,s-s. It is known that several E. coli enzymes require K + for activity 9. For example, several K +requiring enzymes are involved in the synthesis of pufines 1° and pyrimidines n. * Present address: Sloan-Kettering Institute for Cancer Research, New York, N.Y. (U.S.A.)
~N
It. L. I(NNI> ~'l d l .
Since we are able t(, easily regulate the intracellular 1,2 concentrati(m \re at,. in a position to study some .f these reactions in ~,i;,~, in an attempt to determine what K'-requiring reaction is growth limiting. The present study is a survey o1 the in ~'iv,() requirement for t,: for the synthesis of a variety of cell components.
MATERIALS AND METHOI)S
Bach,rial strains, media and growth conditions E. coli strain B2o 7 and B. subtilis 168 K\V mutants defective in their ability to concentrate and accunmlate K ¢ from the growth medium were used ]'3'12. The cells were grown in K ~-containing nmdium A la, supplemented with glucose (o.25 tl~,) and histidine, leucine and methiolfine (IOOktg/ml each) in the case of B2o7, and in the same medium supplemented with 0.2 ° o casamino acids and thymine and tryptophan (20/~g/'ml each) for 14. s~tbtilis. In medimn lacking K~, the potassium phosphates were replaced by an equinlolar concentration of sodium phosphates. All cultures were grown at 37 ~'C with vigorous aeration and were used at approximatel 3 5 "IOS cells per nil. Unless otherwise indicated all media contained adenine (or adenosine), guanine (or guanosine), cytidine and uracil at 20 #g/ml each. Growing cells were centrifuged at 3o00 x g in a Sorvall SP Table Top centrifuge at room temperature, and were washed free of K ~ by two successive resuspensions and sedimentations. The cells were then resuspended it] the appropriate medium indicated ill each experiment.
Determination o/ the synthesis o/ cell components The synthesis of RNA ~, protein 4, DNA 14, murein la, acid-soluble nucleotides t6 and lipid lr were determined as previously described. Growth was determined by increase in turbidity using a Klett-Summerson photoelectric colorimeter (No. 42 filter), ioo Klett units corresponds to 5 ' I°8 cells per ml.
Radioisotopes [2J4C]Uracil (50.3 Ci/mole), [8-1aC_]adenine (52 Ci/nlole), I.iU-14C]leucine (202 Ci/mole), DLU-14C]glucose (I5.5Ci/mole), L-~UJ4C]alanine (I28Ci/mole), and [2-14Cithymine (28 el/mole) were purchased from New England Nuclear Corp., Boston, Mass.
RESULTS
EHect o / K + depletion on growth and synthesis o/cell components The requirement for K+ for growth and the synthesis of a variety of cell components is presented in Fig. I. K+ is required for the growth of E. coli strain B2o 7 (Fig. IA). In the absence of K +, there is practically no increase in absorbance. Similarly, protein synthesis is almost completely inhibited in the absence of K + (Fig. IB). These two findings have been well documented previously 1'~ and are presented here only for heuristic purposes. The synthesis of acid-soluble nucleotides is severely inhibited in cells depleted of K + (Fig. IC). That this is not a function of the lack of protein synthesis in the absence of K + is shown by the observation that
K + REQUIREMENT FOR in vivo SYNTHESIS 3OO
89 4
GROWTH
e~
ACID-SOLUBLE
'£ z5
NUCLEOTIDES
C 2OO
GROWING"
~ ~- U
5
OROW,NG
~,
GROWING
15
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,o
30
60
90
,o
30
o
MINUTES AFTER ADDITION OF [14C]LEUCINE
120
0
MINUTES
20
15 30 45 CoO MINUTES AFTER ADDITION OF [14C'1GLUCOSE
DNA
I-
40 TOTALLIPIDS
~ "x 15
F
GROW:,N
30
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Zw "~0 0~L u
5
0 0 NO K+ 0
15 30 45 60 MINUTES AFTER ADDITION OF [14C]GLUCOSE
2o
Io/,I-// D
15 30 4.5 60 MINUTES AFTER ADDITION OF [14C~THYMINE
O
,
~
J
,
15 30 45 60 MINUTES AFTER ADDITION OF [14C']ALANINE
Fig. i. Requirement for K+ for growth and synthesis of cell components. Exponentially growing cells were depleted of K+ as described in Materials and Methods. All methods for measurements and determinations are described in Materials and Methods. Where indicated chloramphenicol (CM) was used at IOO/~g/ml. Incorporation of [*4C]glucose into acid-soluble nucleotides was determined as previously described*t The cells were incubated under the indicated conditions in the presence of 2 mM [z~C]glucose. At the indicated times i-ml aliquots were added to 2 ml cold 7.5 % perchloric acid. After 60 rain standing in the cold the precipitate was collected by centrifugation and was washed once with 2 ml cold 5 % perchloric acid. The supernatant and wash were combined and o.2 ml 15 ~o Norit was added. The Norit was collected by filtration through Millipore filters (0.45 #m pore size), washed with cold 5 % perchloric acid, dried, and counted in a Beckman Liquid Scintillation spectrometer in Omnifluor. the rate of acid-soluble nucleotide synthesis b y cells i n c u b a t e d in the presence of K ÷ a n d chloramphenicol is initially almost n o r m a l (Fig. IC). At this c o n c e n t r a t i o n chloramphenicol almost completely inhibits protein synthesis a n d growth. T o t a l lipid synthesis is severely depressed in cells i n c u b a t e d in the absence of K + (Fig. ID). These results were also o b t a i n e d using incorporation of ~ P as an i n d e p e n d e n t measure of lipid synthesis. Lipid synthesis continues in cells i n c u b a t e d with c h l o r a m phenicol, although at a somewhat diminished rate compared to growing cells (Fig ID). D N A synthesis (Fig. I E ) in the absence of K + proceeds at a b o u t one-half the rate observed in e x p o n e n t i a l l y - g r o w i n g cells (see also refs. 8 a n d 14). Murein synthesis continues b u t at a slower rate t h a n n o r m a l in K+-depleted cells for a b o u t 30 m i n a n d t h e n a b r u p t l y stops (Fig. I F ) .
{)0
H. 1.. I';NNIS ' / ~t/.
I)ltriJzcs a~zd pyrimidim:s stimztlah~ the s_wzthcsis o~ RA'A in l£ -dc/)k,&d cc/l,s RNA synthesis continues in cells depleted of K (see refs 4 and (~ 8). How
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O
15 30 45 60 MINUTES AFTER ADDITION OF [14C]ADENINE
Fig. 2. Stimulation b y purines a n d pyrimidines of R N A synthesis in E. coil in the absence of K+. (A) Growing cells of strain B2o 7 were depleted of K~ as described in Materials and Methods. [t4C]Uracil (o. 3 ~Ci/mI; io/~g/ml) was added and samples were t a k e n at the indicated intervals. I n c o r p o r a t i o n of [14C]uracil into R N A was carried o u t as described in Materials and Methods. A growing culture was similarly handled. 2o Mg/ml non-radioactive adenine or guanine were used where indicated. (B) This e x p e r i m e n t was the same as outlined in (A) except t h a t [14C]adenine (o. 3/~Ci/ml; i o / z g / m l ) and 2o/*g/ml non-radioactive uracil were used. K+-depleted cells plus uracil (@) ; K+-depleted cells, no uracil ( • ) .
91
K + REQUIREMENT FOR in vivo SYNTHESIS
That uracil is also required for maximum stimulation of RNA synthesis in K+-depleted cells is shown in Fig. 2B. In this experiment the incorporation of [z4C] adenine into RNA was determined in the presence and absence of non-radioactive uracil. As can be seen, the presence of uracil greatly stimulated the incorporation of [l*C]adenine. As seen in Fig. 3, purines and pyrimidines are also required for maximum stimulation of RNA synthesis during K+ depletion of a B. subtilis mutant defective in K ÷ accumulation. In this mutant, adenosine (or adenine or inosine) alone stimulates the incorporation of [14C]uracil much better than guanosine (or guanine) alone. The addition of both adenosine and guanosine gave no more stimulation than observed using adenosine alone (data not shown). We previously showed that RNA synthesis proceeded during bacteriophage T 4 infection of E. coli B2o 7 in the absence of K ÷ (see ref. 18). The data presented in Fig. 4 show that added purines and pyrimidines stimulate the synthesis of RNA in cells infected in the absence of K ÷ over 3-fold that observed in the absence of compounds. No significant stimulation by added purines and pyrimidines of RNA synthesis during infection in the presence of K ÷ was noted. 75
150
RNA
/
RNA E_.COLI /
p
T4-1NFECTED
~
'---" " 45
90
~
u
POTASS
~ a
~--~ 60 u ~ 0u
/
8 6O
12o
"--' ..m Z =E
/
~
Lu30
~ m
3O
I
Z I
0
15
30
45
60
I
I
J
15
30
45
MINUTES AFTER ADDITION
MINUTES AFTER ADDITION
OF [14C]URACIL
OF [14C] URACIL
Fig. 3. Stimulation b y p u r i n e s a n d pyrimidines of R N A synthesis in B. subtilis in t h e absence of K +. This e x p e r i m e n t was p e r f o r m e d as outlined in Fig. 2A, u s i n g B. subtilis s t r a i n K W . Fig. 4. S t i m u l a t i o n b y p u r i n e s and pyrimidines of R N A synthesis in E. coli infected w i t h bacteriophage T 4 in the absence of K+. Growing cells or K+-depleted cells of s t r a i n B2o 7 were infected w i t h bacteriophage T 4 a t a multiplicity of 5 bacteriophage per cell as previously described 31. Less t h a n i % of the cells infected survived. I n c o r p o r a t i o n of [14C]uracil into R N A (exclusive of bacteriophage DNA) was determined as previously described St.
DISCUSSION
By use of mutants of E. coli and B. subtilis defective in their ability to accumulate K+ from the growth medium, we have been able to study the in vivo requirement
02
H. 1.. I. NNIS ,'/ ~g/.
for this cation for the synthesis of a variety of cells components. K depletion is a simple and rapid procedure. Cells are viable for long periods of time in the ab>ence of K " (see ret. 8). ]'lie unique feature of this m e t h o d (as shown by previous w,,rk a) is t h a t one can maintain a very fine control over the intracellular K ~ concentration merely by the simple manipulation of the anlount of K I in the growth nledium. To our knowledge this is the only method available for this purpose. One need only add back K - to the depleted cell culture to reiniate growth. Since it is kn,~wn that several enzymes require K~ for activity or are stimulated by the cation we were in a position to s t u d y these reactions, anti others, i~, vivo. We had previously shown t h a t K ¢ was required for growth and protein swlthesis *'4. In the present investigation we further delnonstrate that K is also required for the synthesis of purines and pyrinfidines. This confirms work done on the K requirement for the i,a vitro activity of certain enzyines involved in purine and pyrinfidine synthesis ~°'n. When purines and pyrimidines are added to cultures of cells depleted of K +, R N A synthesis is stimulated by approximately 4-6 fold t h a t observed in depleted cells lacking the bases. Consequently, although K ~ has been implicated in tim M vitro formation of G T P and d G T P fronl G D P and d G D P respectively l'q, it is obvious t h a t enough GTP is made i,z vivo, perhaps by an alternative lmthway, to supply some of the G T P and d G T P needed for RNA and D N A synthesis, resl)ectively. D N A synthesis in the absence of K+ proceeds at about one-half the initial rate compared to growing cells, (see also ref. i4). Murein synthesis also proceeds in the absence of K+ but only to a limited extent. More importantly, total lipid synthesis is ahnost completely inhibited in the absence of K +. The consequences of this defect on cell metabolisin is not known. This observation is in agreement with previous work which showed t h a t K r (but not Na +) was needed for CDP-diglyeeride formation in microorganisms '0 "2. Perhaps K~ is also required for an earlier step in lipid synthesis. K - has been reported to be necessary for the in vitro activity of ribonuclease I I 2a "~" of E. coll. This enzyme m a y be involved in messenger R N A decay in vivo as well as i~a vitro and consequently m a y have important regulatory significance "a,27 a0. This can be studied by use of the K+-dependent n m t a n t s with the aim of stabilizing messenger R N A under conditions of K + depletion. Since we have already shown that K + is required for bacteriophage T 4 messenger R N A breakdown is, we are now studying the role of K + in E. coli messenger R N A nletabolism.
REFERENCES M. L u b i n a n d D. Kessel, t3iochem. Biophys. Res. Commun., 2 (196o) 249. W. E p s t e i n a n d B. S. Kim, .[. Bactcriol., i o 8 (1971) 639. M. L u b i n , Fed. Proc., 23 (1964) 994. M. L u b i n a n d H. L. Ennis, Biochim. Biophys. dcta, 80 (1964) 614. G. L ester, J. BacterioL, 75 (1958) 426. H. L. E n n i s a n d M. L u b i n , Biochim. Biophys. Acta, 95 (1965) 605. H. L. E n n i s a n d M. L u b i n , Biochim. Biophys. Acta, 95 (I965) 624. H. L. E n n i s a n d M. L u b i n , Biochim. Biophys. Acta, 5o (1961) 399. C. H. Seulter, Science, 168 (197 o) 789. J. M. B u c h a n a n , 7"he Harvey Lectures, 1958-1959, Series LIV , Acadenfic Press, N e w Y o r k , I96O, p. lO4. I I P. M. A n d e r s o n a n d A. Meister, Biochemistry, 5 (196o) 3157 . I 2 3 4 5 6 7 8 9 io
K + REQUIREMENT FOR in viuo SYNTHESIS 12 13 14 15 16 17 18 19 2o 21 22 23 24 25 26 27 28 29 3°
93
D. B. Willis and H. L. Ennis, J. Virol, 3 (1969) i. B. D. Davis and E. S. Mingioli, J. Bacteriol,, 6o (195 o) 17. K. E. Rubenstein, E. Streibel, S. Massey, L. Lapi and S. S. Cohen, J. Bacteriol., in the press. E. J. J. Lugtenberg and P. G. De Haan, Antonie van Leeuwenhoek, J. Microbiol. Serol., 37 (1971) 537. Y. Sokawa, E. Nakao-Sato and Y. Kaziro, Biochim. Biophys. Acta, 199 (197 o) 256. J. Kanfer and E. P. Kennedy, J. Biol. Chem., 239 (1964) 172o. P. S. Cohen and H. L. Ennis, Virology, 27 (1965) 282. M. P. Oeschger and M. J. Bessman, J. Biol. Chem., 241 (1966) 5452. J. t{. Carter, J. Lipid Res., 9 (1968) 748. R. E. McCaman and W. R. Finnerty, J. Biol. Chem., 243 (1968) 5o74. P. H. Patterson and W. J. Lennarz, J. Biol. Chem., 246 (1971) lO62. P. F. Spahr, J . Biol. Chem., 239 (1964) 3716. P. F. Spahr and D. Schlessinger, J. Biol. Chem., 238 (1963) PC225I. M. F. Singer and G. Tolbert, Science, 145 (1969) 593M. F. Singer and G. Tolbert, Biochemistry, 4 (1965) 1319. T. Kivity-Vogel and D. Elson, Biochem. Biophys. Res. Commun., 33 (1968) 412. M. Artman, N. Silman and H. Engelberg, Biochem. J., lO 4 (1967) 878. H. O. Voorma and L. Bosch, Biochem. Biophys. Res. Commun., 18 (1965) 42o. E. T. Lennette, L. Gorelic and D. Apirion, Proc. Natl. Acad. Sci. U.S., 68 (1971) 314o..