Tim JOURNAL OF BIOLOQICAL CAEMISTRY Vol.246, No. 14,Issue of July 25, pp. 4381-4385, 1971

Printed in U.S.A.

On the Regulation of Guanosine Tetraphosphate Levels in

Stringent and Relaxed Strains of Escherichia coli

(Received for publication, January 25, 1971)

ROBERT A. LAZZARINI AND MICHAEL CASHEL

From the Laboratory of Molecular Biology, National Institute of Neurological Diseases ancl Stroke, il’ational Institutes of Health, Public Health Service, TJnited States Department of Health, Education, and Weuare, Be- thesda, Maryland 2’0014

JONATHAN GALLANT

From the Department of Genetics, University of Washington, Seattle, Washington 98105

SUMMARY

We have examined the intracehular levels of the guanosine tetraphosphate, guanosine 5’-diphosphate 2’- or 3’-diphos- phate (ppGpp), in an isogenic pair of stringent and relaxed strains of Escherichia coli. As previously observed, relaxed strains do not accumulate ppGpp during amino acid depriva- tion, while stringent strains do. However, under all other culture conditions examined, relaxed and stringent cells regulate their ppGpp levels similarly. During carbon dep- rivation or step-down transitions both strains accumulate ppGpp. Neither strain accumulates appreciable amounts during phosphate deprivation or after treatment with colicin El. The basal lev*ls of ppGpp observed in both stringent and relaxed strains during balanced growth are, in most cases, the same, and vary inversely with the growth rate and RNA content of the cells.

When Escl~erichia coli cells are deprived of an essential amino acid or arc unable to activate one, there is a severe and pref- erential restriction of ribosomal and transfer RNA synthesis (l-5). This major adjustment of biosynthetic activity which serves to maintain the RNA to protein ratio of the cell is termed stringent control of RNA synthesis. This control requires t,he product of the rel gene: rel- i strains are unable to curtail ribo- somal RNA synthesis and maintain the RNA to protein ratio of the cell during amino acid deprivation (6-8). Within a few seconds of the imposition of amino acid deprivation, relf cells begin to accumulate an unusual tetraphosphate of guanosine, guanosine ii’-diphosphate 2’- or 3’-diphosphate (pp\+p) above the basal levels found in growing cells (9, 10). Although a basal level of ppGpp is demonstratable in rel- cells (ll), this level does not increase appreciably during amino acid deprivation.

The coincidence of high intracellular ppGpp levels and the failure of cells to accumulate RNA during amino acid depriva-

* The abbreviat,ions used are: rel-, relaxed control; leE+, strin- gent control.

tion and the kinetics with which changes in ppGpp levels are effected by the cell suggests that this compound participates in the amino acid control of RNA synthesis (9). Direct support for the causal role of ppGpp in the inhibition of ribosomal RNA synthesis was obtained by Travers, Kamen, Schleif, and Cnshel (12, 13). They found that ribosomal RNA synthesis required, in addition to the core RNA polymerase and u factor, a protein factor, $r. They further showed that the #r-mediated stimu- lation of ribosomal RNA synthesis was preferentially inhibited

by PPGPP. Under conditions other than amino acid deprivation, both

relaxed and stringent cells are able to regulate the rate of ribo- somal RNA synthesis and adjust the RNA:DNA ratio of the cell (7). When transferred from a rich medium to a poor one, both relaxed and stringent cells sharply restrict their synthesis of ribosomal RNA while allowing continued protein accumula- tion (14). When the RNA to protein ratio characteristic of the new medium is achieved, ribosomal RNA synthesis resumes at a rate characteristic of cells cultured on the poorer medium. The mechanism controlling RNA synthesis during growth rate tran- sitions is not understood at present. However, the fact that it shares several characteristics with that operating during amino acid starvation of a stringent strain suggests that they may be related (14-16).

In the present communication we show that during growth rate transitions both relaxed and stringent cells accumulate ppGpp in amounts approaching those observed during the strin- gent response to amino acid starvation. Similarly, during a carbon source deprivation both relaxed and stringent cells ac- cumulate large amounts of ppGpp. The basal levels of ppGpp found in relaxed and stringent cells during balanced growth are, in most cases, the same and vary inversely with growth rate and RNA content of the cells. On the basis of these observations we suggest that the intracellular ppGpp levels and the rate of ribosomal RNA synthesis are interrelated.

EXPERIMENTAL PROCEDURE

Bacteria and Culture Conditions-E. coli NFl61 (filet, arg, reZ+) and NE’162 (met, urg, rel-) were obtained front Dr. Nils Fiil. These strains are of the K-12 lineage and arc believed to

4381

by guest on August 25, 2015

http://ww

w.jbc.org/

Dow

nloaded from

4382 Vol. 246, No. 14

be isogcnic except for a small region of the chromosome which iuclutlcs the rel locus. E. coli P687-54 (‘01 E1 (17) was obtained fro111 1)r. John Inselburg. Bacteria were cultured with the Tris minimal medium described by Kaempfer and Magasanik (18), except that the KII~l’Ol concentration was 10e3 M. The carbon sources for the media were glucose (0.25c/,), succinate (0.3%), or alanine (0.50;). Minimal media contained 50 pg per ml of each required amino acid. Where noted in the text, minimal medium was supplemented with 0.47;) vitamin-free Casamino a&Is. Bacteria were cultured with reciprocal shaking in a water bath at 37”. Growth was measured by following the ab- sorbance at 600 nm with a Zeiss PMQ II spectrophotometer. 1Jntlcr the conditions used, a cell concentration of 5 X lo* cells per 1111 yielded a turbidity of 1 at 600 Tim.

Determination of Intracellular ppGpp Levels-The ppGpp lev- cls ill [Y’] phosphate labeled cells were determined radiochemi- cajly after extraction of the acid-soluble pools with formic acid and i;epnrntion of ppGpp by thin layer chromatography (9). In :I typical csl)crimcnt, cells were cultured in the requisite metlium to a density of 0.5 x lo8 cells per ml and [32P]phos- phoric acid added (final specific activity 200 to 500 &i per pmole) to a portion (1 tb 2 ml) of the culture. Growth was nlolritored in the unlabeled portion of the culture.

When high levels of ppGpp were expected, a one-dimensional separation with 1.25 or 1.5 M KIM’Ol as the developing solution (9, 19) was used. When basal or very low levels of ppGpp were mc:lsured, a two-dimensional separation was used to reduce the background radioactivity. The chromatograms were developed with the step-formate system in the first dimension and with I .25 M KR&‘O~ in the second (19). ppGpp was located by radioautography and its radio content determined by liquid scintillation counting.

The [32P]-phosphate specific activity of the media was deter- mined for each individual experiment. A portion (5 ~1) of the suitably diluted sample of medium was spotted on a circle of polycthyleneiminc cellulose, dried, and counted in Liquiflor scintillation fluid. The phosphate concentration of the medium at the tirne of the [Y’]phosphate addition was assumed to be the ~amc as that of fresh media (lob3 M), although the bacteria had been cultured in it to a density of approximately 0.5 x lo* cells per ml. The error introduced by this assumption is approxi- mately a 55% overestimation. In the case of rnedia that con- tained Casamino acids, the phosphate concentration at the time of [92P]phosphate addition was determined with the calorimetric assay of Arnes and Dubin (20).

Calorimetric Rstimation of RNA and DNA-To determine the RXA and DNA content of cells, duplicate samples (25 ml) were removed from a growing culture and rnixed with 2.5 ml of 500/, trichloroacetic acid at 0”. After at least 15 rnin at 0” the cells were collected on glass fiber filters and washed with 45 ml of 5% trichloroacetic acid. The filters containing the entrapped cells were folded and placed in the bottom of a test tube, 13 x 100 mm, and covered with 2 ml of trichloroacetic acid (5yi). The tube was cal)pcd with a marble and placed in a 90-95” wat,er bath for 20 mill. After cooling the tube, the glass fibers were sedimentecl by centrifugation and portions of the supernatant fluid were used in either the orcinol calorimetric assay for RNA (21) or the di- phenylamine assay for DN.4 (22). When only RNA was to be determined the initial volume of cells removed from t,he culture was reduced to 5 ml.

Preparation of Colicin K,-The colicinogenic strain P687-54

was grown in Tris glucose rnedium supplemented with Casamino acids to a density of 2 x lo* cells per ml. Colicin production was induced with 0.2 kg per ml of mitomycin, as described by Maeda and Nornura (23). After induction, the cells were harvested by centrifugation, washed twice, and ruptured by passage through a French pressure cell, as described by Fields and Luria (24). The extract was clarified by centrifugation at 105,000 x g for 30 min and frozen in small portions. The colicin titer (2 x 1Ol2 per ml) was determined from the fraction of cells surviving exposure to the colicin preparation, with the equation b/b,, = e-“, where b and bo are the number of viable bacteria after and before exposure to the colicin preparation and m is the multiplicity. In all cases the test organism was E. coli NF161. Bacterial titers were deter- mined by plating sufficiently diluted samples on nutrient broth agar plates.

Materials--Silicate-free 32P-orthophosphoric acid was obtained from Tracerlab, Division- of Laboratory for Electronics, Inc. I’olyethylcncimine cellulose sheets (MN-polygram cell 300 poly- ethylenc~iminc) were the product of Brinkman Instruments, Inc. Nitroccllulosc filters (Metricel, GA-6, 47 mm diameter, 0.45 ~1 pore size) and glass filters (25 rnm diameter, type A, withoutj binder) were purchased from Gelman Instrurnent (‘ompany. Liquiflor was a product of New England Nuclear.

RE:SULTS

Accumdation oj ppGpp during Amino Acid Deprivation-In a

previous publication ppGpp was shown to accumulate in strin- gent strains but not in relaxed strains during amino acid depriva- tion (9). In the present report we show the accumulation of ppGpp in relaxed as well as stringent strains under several condi- tions of nutritional deprivation. For the purposes of compari- son, the changes in the intracellular concentration of ppGpp in the isogenic pair NF161 and NFl62 during methionine depriva- tion are presented in Fig. 1. Upon exhaustion of the methionine in the rnedium the stringent strain abruptly accumulates large amounts of ppGpp above the basal level of 0.04 to 0.05 nmole per AGoo norrnally found in this strain. In contrast the relaxed strain, NF162, after amino acid deprivation shows no detectable

TIME (hours)

FIG. 1. Accumulation of ppGpp by Escherichia coli during methionine deprivation. At zero time growing cultures of E. coli NF161 and NF162 were filtered, washed, and resuspended in fresh medium containing 3 pg per ml of methionine. [32P]PO( (400 &i per ml) was added to a l-ml portion of each culture. Growth (- - -) was monitored turbidimetrically in the unlabeled culture while ppGpp accumulation (--) was measured in aliquots in the labeled culture.

by guest on August 25, 2015

http://ww

w.jbc.org/

Dow

nloaded from

Issue of July 25, 1071 R. A. LaxxariG, IV. Cashel, and J. Gallant 4383

TABLE I

ppGpp levels in E. coli NF162 during single and multiple amino

acid deprivations A growing culture of E. coli NF162 was filtered and resllspcndcd

in complete medium which was lacking amino acids. Portions of the culture were either left unsupplemented, or supplemented with arginine alone, methionine alone, or arginine and methionine

together. [32P]orthophosphate (500 ,uCi per pmole) was added to each culture, and samples were removed for ppGpp determinations after 30 min.

Labeling condition

Complete.. / - Methionine. . . . . . - Arginine. . - Methionine and arginine. . .

14 14.6 12.1 39.4

TIME (hours)

increase above its basal level of 0.01 to 0.02 nmole per AeoO. The failure of NF162 to increase its ppGpp level cannot be attributed to a lack of severity of the amino acid starvation, since depriva- tion of arginine alone or simultaneously with methionine only marginally increases the intracellular concentration of ppGpp (Table I).

FIG. 2. Accumulation of ppGpp by Escherichia coli NFlGl md

NF162 during glucose deprivation. At zero time growing cultures of each organism were filtered, washed, and resuspended in fresh medium containing 0.025% glucose. [“ZP]P04 (200 pCi per ml) was added to l-ml portions of each culture. Growth (- - -) and ppGpp accumulation (---) were monitored as in Fig. 1.

500, I I I I I I I I I I

Accumulation of ppGpp during Carbon LIeprivation and Step- Down Transitions-The ability of relaxed strains to accumulate substantial amounts of ppGpp can be shown during a simple carbon source starvation. In Fig. 2 the effects of glucose starva- tion on the ppGpp levels of NF161 and NF162 are shown. The stringent strain, NF161, upon carbon deprivation accumulates amounts of ppGpp that are equivalent to those found in amino acid-deprived cells. The relaxed counterpart, NF162, also ac- cumulates ppGpp upon carbon deprivation, but at a slower rate: after 1 hour of deprivation the level of ppGpp in the relaxed strain is approximately one-half that found in t,he stringent strain after 10 min of deprivation. Qualitatively, the same results can be observed during a step-down transition between a rich and a poor medium. In the example shown in Fig. 3, NF161 and NF162 exhaust the glucose present in the medium at a cell density of 2 x lo8 and are forced to use succinate that is also present in the medium as a source of carbon. As pointed out by Niedhardt (7), both cultures traverse the step-down transition in a nearly identical manner. After depleting the glucose both strains enter a diauxic lag during which the turbidity of the cultures slowly in- creases, presumably as the result of the synthesis and accumula- tion of protein and DNA. RNA accumulation abruptly ceases at the break in the growth curve and does not resume until the turbidity of the culture increases 1.35.fold above that observed at glucose depletion.2 During this period the RNh:DNA ratio of the cells is adjusted to that characteristic of cells cultured on the poorer medium (15). In the present case the RNb1:DNA ratio changes from 7.4 to 5.5 (see below and Fig. 5).

TIME (hours)

FIG. 3. Accumulation of ppGpp by Escherichia coli NFl61 and NF162 during a glucose to succinate step-down kansition. 1Xach strain of E. coli was cultured in Tris minimal medium containing 0.025% glucose and 0.394 succinate as sole carbon sources. At zero time [32P]POd was added to l-ml portions of each culture to yield a final concentration of 100 &i per pmole. Growth (-- - -) and ppGpp accumulation (--) were monitored during the collrse of the step-down transition as in Fig. 1.

Upon depletion of the glucose both strains behave as they do during a simple glucose deprivation and accumulate ppGpp, al- though a utilizable carbon source is present in the media. The difference in the kinetics of appearance of ppGpp in the two strains during the carbon deprivation or step-down transition might in fact be related to an endogenous amino acid deprivation

that is occasioned by the eshaustion of the easily utilizable glu-

case. Thus, the rapid accumulation of ppGpp in NFl61 rn:ly re- flect a response to amino acid starration, superimposed on c::wbon

source deprivation. The relased strain being insensitive to amino acid deprivation however would synthesize ppGp1) ill re-

sponse to only one stimulus. Furthermore, the difference in rate of ppGpp accumulation apparent in Fig. 3 is not observed in all step-down transitions. With another relaxed and stringent pair of strains, E. coli EAl and EA2, a carbon source step-down from glucose to proline elicits approximately the same rate of ppGpp accumulation in both strains, although the stringent strain cren- tually accumulates slightly more. In still another isogenic pair of E. coli strains, CP78 and CP79, undergoing a step-down from glucose to lactate, ppGpp accumulates in both strains at approxi- mately the same rate.3 In this latter case, the final lerel of ppGpp in both strains is considerably less (0.2 nmole per AGO,,)

e R,. A. Lazzarini, unpublished experiments. 3 I<. M. Winslow, unpublished experiments.

by guest on August 25, 2015

http://ww

w.jbc.org/

Dow

nloaded from

4384 Regulation o;f ppGpp Levels Vol. 246, No. 14

than is observed in NF161 undergoing the glucose to succinate shift.

Limited ppGpp Accumulation during Restricted Phosphate or Nucleotide Metabolism-Several experimental or nutritional con- ditions which restrict RNA accumulation presumably by altering the availability of nucleotide triphosphates elicit little or no ppGpp accumulation. Phosphate deprivation of NF161 and NF162 leads to only a marginal increase in the ppGpp level of both strains (Fig. 4). Upon depletion of phosphate in the me- dium the turbidity of the cultures continue to increase but at a much reduced rate. Within 5 min of the break in the growth curve the major purine nucleotide pools, ATP and GTP, contract

TIME (hours)

Fro. 4. Accumulation of ppGpp by Escherichia coli NF162 and NF161 during phosphate deprivation. Growing cultures of both strains of E. coli were filtered and resuspended in fresh media containing 1 X 10-4 M phosphate. [32P]P0, was added to portions of each culture to yield a final concentration of 40 pCi per ml. Growth (- - -) and ppGpp accumulation (-) were monitored as in Fig. 1.

0 L ’ I I ,\ j 0 4 6 8 IO 12

RNA/DNA

FIG. 5. Basal levels of ppGpp and RNA content of Escherichia coli cultured on different growth media. E. coli NF161 (0) and NF162 (A) were cultured on minimal media containing as a sole carbon source 0.5% alanine, 0.3% succinate, 0.25% glucose or 0.25y0 glucose plus 0.40/, Casamino acids. The cells were cultured for four to six doublings in the appropriate medium prior to the addition of [3zP]POc (400 &i per ml). After one doubling in the presence of [32P]P0, samples were removed for ppGpp estima- tion; RNA and DNA were determined in the same sample of a parallel unlabeled culture. The doubling times for both E. coli NF161 and NFl62 were 205 min with alanine, 90 min with suc- einate, 55 min with glucose, and 40 min with glucose plus Casamino acids as sole sources of carbon.

to approximately one-third of their normal size but do not un- dergo any further change. The int.racellular concentration of ppGpp, on the other hand, first expands slightly, and then con- tracts upon continued phosphate deprivation.

Neither uracil nor guanine deprivation of stringent auxotrophs of E. coli elicits ppGpp accumulation. Treatment of E. coli with colicin El leads to the rapid inhibition of RNA, protein, and DNA syntheses (25). It has been proposed that the inhibition of these diverse synthetic processes is a secondary effect of a pri- mary inhibition of oxidative phosphorylation (25). Treatment of NF161 and NF162 with colicin preparations at a multiplicity of 5 leads to an abrupt reduction in the intracellular levels of ATP and GTP but less than a 2-fold increase in the intracellular ppGpp concentration.

Intracellular Levels of ppGpp during Balanced Growth-Under all of the experimental and nutritional conditions examined ex- cept amino acid deprivation, relaxed and stringent cells similarly regulate their intracellular ppGpp levels. We were led to in- vestigate whether the similarity in the control of ppGpp levels extended to cells in balanced growth as well. For this purpose, ppGpp levels were measured in relaxed and stringent cells grow- ing on several media which support growth at widely different rates. Since the RNA content of a cell varies regularly with growth rate (26,27), this comparison also can reveal a systematic variation of ppGpp level with RNA content. The results, shown in Fig. 5, indicate that relaxed and stringent cells contain ap- proximately the same level of ppGpp when cultured in the same medium, except for glucose minimal medium. In that case the relaxed contains about one-third the ppGpp level of the strin- gent (11). Furthermore, it is clear that with the same exception, the levels of ppGpp vary regularly and approximately linearly with the RNA content of the cell.

DISCUSSION

The nucleotide ppGpp was originally observed as material which accumulated in stringent but not in relaxed cells during amino acid deprivation (9). The present observations provide a more comprehensive picture of the conditions governing the accumulation of ppGpp. It is clear that relaxed cells are by no means unable to produce the nucleotide: relaxed cells contain a significant basal level of ppGpp, which varies with growth rate, and they are able to accumulate r&her high levels during carbon source starvation or carbon source downshift. We may there- fore reject the simplest interpretation of earlier results, namely that the rel- mutation inactivates the enzyme responsible for ppGpp biosynthesis. The rel- mutation seems rather to affect the relationship between ppGpp formation and some specific con- sequence of amino acid deprivation. Since relaxed mutants do accumulate ppGpp during downshift, but do not during amino acid deprivation, it follows that these two nutritional conditions regulate the nucleotide’s accumulation through different mecha- nisms, or by means of different signals.

Neidhardt (7) has reported that several different stringent and relaxed strains show a similar variation in RNA content with growth rate. The RNA:DNA ratios presented in Fig. 5 extend this conclusion to an isogenic pair of strains, confirming the con- clusion that “growth rate” control of RNA accumulation is un- impaired by the rel- mutation. The basal levels of ppGpp also vary systematically with growth rate in both members of the iso- genie pair, leading to a striking inverse correlation between ppGpp concentration and RNA:DNA ratio (Fig. 5). I f the

by guest on August 25, 2015

http://ww

w.jbc.org/

Dow

nloaded from

lssuc of *July 25, 1971 R. A. Laxzarini, M. Cashel, and J. Gallant 4385

line:lr relationship displayed in Fig. 5 extends to very low levels of ppC;pp, one would expect a limiting RNA : DNA ratio of about 11, B. coli cells cultured on nutrient broth, at near maximum growth rate, have an RNA:DNA ratio of 10. The one departure from this rqulnr relationship, that of relaxed cells growing in glucose minimal Incdium, is at present an enigma.

There seem to be at least two physiological mechanisms by which net synthesis of RNd is curtailed. During amino acid deprivation of a stringent strain (4), and during carbon source downshift of a relaxed (presumably also of a stringent) (14), the synthesis of ribosomal and transfer RNA is preferentially in- hibited. On the other hand, during deprivation of uracil (28) or phosphate (29, 30), ribosomal RNA appears to be made, but fails to accumulate because synthesis is offset by abnormally rapid degradation. At present, there is not sufficient evidence available to permit conclusions as to the mode of RNA control exercised during guanine deprivation, colicin treatment, or bal- anced growth at different rates. Nonetheless, the rudimentary correlation that emerges from this comparison is that ppGpp accumulates under two conditions where ribosomal RNA synthe- sis is preferentially inhibited, while under at least two other con- ditions RNA accumulation is restricted by rapid degradation and ppGpp does not accumulate. This correlation suggests that the control of ribosomal RN.4 synthesis involves pl)Gpp while the control of ribosomnl RNB degradation does not.

Recent studies on transcription in vitro suggest a plausible mechanism for the preferential inhibition of ribosomnl RNA syn- thesis by ppGpp. Cashel (31) has shown that ppGpp inhibits core and u-supplemented RNA polymerase in a specific although limited manner. Moreover, Travers, et al. (13) have shown that ppGpp preferentially inhibits the production of ribosomal RNA in vi&o by RNA polymerase in the presence of #r factor. These observations suggest that ppGpp governs the specificity of RNA polymerase, and in particular the ability of the enzyme plus $r factor to transcribe the ribosomal RNA cistrons. Since the RNA content of cells reflects predominantly the quantity of ribosomal RNA, the relationship shown in Fig. 5 is consistent with this model.

Our results are also consistent with a different interpretation of the relationship between ppGpp and RNA synthesis. Wong and Nazar have observed that the addition of rifampicin to cells which have accumulated ppGpp during amino acid deprivation (32) results in a reduction in ppGpp concentration, which appears to precede an effect on protein synthesis. The eventual disap- pearance of ppGpp after the inhibition of protein synthesis is to be expected, since inhibitors of protein synthesis antagonize the stringent response (33). These results suggest that the RNA polymerase itself may be responsible for the synthesis of ppGpp and that the accumulation of large amounts of ppGpp may be a consequence rather than a cause of the restricted RNA accumu- lation. However, the significance of the rifampicin effect is diffi- cult to assess, since the effect may be an indirect one. Experi- ments to be reported elsewhere4 will show ppGpp formation can proceed in the presence of rifampicin under certain conditions, suggesting that the effect of rifampicin on ppGpp accumulation is indirect.

4 H. Erlich, T. Laffler, and J. Gallant, unpublished experiments.

1.

2.

3.

4.

5.

6.

7. 8. 9.

10.

11.

12.

13.

14.

15.

16.

17. 18.

19.

20. 21. 22. 23. 24. 25. 26.

27.

28.

29.

30.

31.

32.

33.

REFERENCES

In summary, the results presented here indicate that relaxed mutants have lost the ability to produce ppGpp in response to amino acid deprivation, but retain the ability to produce the nucleotide in response to other conditions. The condit’ions under which ppGpp accumulates suggest that it participates in a mech- anism governing the synthesis of ribosomal RXA in both strin- gent and relased cells.

STENT, G. S., AND BRENNER, S., Proc. Nat. Acacl. Xci. U. S. A., 47, 2005 (1!161).

FANGMAN, W. L., AND NEIDH.~RDT, F. C., J. Biol. Chem., 239, 1844 (1964).

FORCHHAMMER, J., AND KJELDGAARD, N. O., J. Mol. Biol., 37, 245 (1968).

LAZZBRINI,. R. -4., AND DAHLBERG, A. E., J. Biol. Chem., 246, 420 (1971).

L~v.YLL~, It., .ZND DE H.~u~EI~, G., J. Mol. Biol., 37, 269 11968’, \ - - - - I

BORF,K, E., R.OCI<F:NBACK, J., AND RYAN, A., J. Bacterial., 71, 318 (1956).

NEIDIIUDT, F. C., Biochim. Biophys. Acta, 68, 365 (1963). NEIDIXAI~D~P, F. C., Biochim. Biophys. Acta, 68, 380 (1963). CASHEEL, M., J. Hiol. Chem., 244, 3133 (1969). CASIIEL, M., AND KALIUCHER, B., J. Biol. Chem., 246, 2309

(1970). GALLANT, J., ERLICH, H., HALL, B., AND LAFFLER, T., Cold

Spring Harbor Symp. Quant. Biol., 36, 397 (1971). TRAVERS, A., KAMEN, R., AND SCHLIEF, R. F., Nature, 228, 748

(1970). TRAVERS, A., KAMEN, R., AND CASHEL, M., Cold Spring Harbor

Symp. Quant. Biol., 36, 415 (1971). LAZZARINI, R. A., AND WINSLOW, R. M., Cold Spring Harbor

Symp. Quant. Biol., 36, 383 (1971). NIXDHARDT, F. C., AND FRAENKEL, D. G., Cold Spring Harbor

Symp. Quant. Biol., 26, 63 (1961). MAAL@, O., Twenty-eighth Symposium of Society for Develop-

mental Biology: Communication in Development, University of Colorado, 1969.

INSELBURG, J., J. Bacterial., 102, 642 (1970). KAEMPFER, R. 0. R., AND MAGASANIK, B., J. Mol. Biol., 27,

475 (1967). CASH&+ M., LAZZAIEINI, R. A., AND KALBACHER, B., J. Chro-

matogr., 40, 103 (1969). AMES, B. N., AND DUBIN, D. T., J. Biol. Chem., 236,769 (1960). ScHNmDk:R, W. C., J. Biol. Chem., 161, 293 (1945). BURTON, K. A., Biochem. J., 62, 315 (1956). MAEDA, A., AND NOMURA, M., J. Bacterial., 91, 685 (1966). FIELDS, K. L., AND LURIA, S. E., J. Bacterial., 97, 57 (1969). FIELDS, K. L., AND LURIA, S. E., J. Bacterial., 97, 64 (1969). NEIDIURDT, F. C., AND MAGASANIK, B., Biochim. Biophys.

Acta, 42, 99 (1960). SCHAECHTICR, M., MAAL$E, O., AND KJELDG~ARD, N. O., J.

Gen. Microbial., 19, 592 (1958). LAZZARINI, R. A., NAKATA, K., AND WINSLOW, It. M., J. Biol.

Chem., 244, 3092 (1969). JULIEN, J., ROSSET, R., AND MONIER, R., Bull. SOC. Chim.

Biol., 49, 131 (1967). HORIUCHI, T., HORIUCHI? S., AND MIZUNO, D., Biochim. Bio-

phys. Acta, 31, 570 (1959). CASH~L, M., Cold Spring Harbor Symp. Quant. Biol., 36, 407

(1971). WONG, J. T.-F., AND NAZAR: R. N.: J. Biol. Chem., 246, 4591

(1970) EZEKIEL, D. H., AND ELKINS, B. N., Biochim. Biophys. Acta,

166, 466 (1968).

by guest on August 25, 2015

http://ww

w.jbc.org/

Dow

nloaded from

Jonathan GallantRobert A. Lazzarini, Michael Cashel and 

Escherichia coliRelaxed Strains of Tetraphosphate Levels in Stringent and On the Regulation of GuanosineBIOCHEMICAL GENETICS:CONTROL MECHANISMS AND

1971, 246:4381-4385.J. Biol. Chem. 

  http://www.jbc.org/content/246/14/4381Access the most updated version of this article at

  .JBC Affinity SitesFind articles, minireviews, Reflections and Classics on similar topics on the

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/246/14/4381.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on August 25, 2015

http://ww

w.jbc.org/

Dow

nloaded from