Vol. 121, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 923-932 Copyright 0 1975 American Society for Microbiology

Strain of Escherichia coli with a Temperature-Sensitive Mutation Affecting Ribosomal Ribonucleic Acid Accumulation1 TERRY FREY,2 LONNIE L. NEWLIN, AND ALAN G. ATHERLY* Department of Genetics, Iowa State University, Ames, Iowa 50010

Received for publication 17 December 1974

A mutant of Escherichia coli has been isolated that has a temperature-sensitive mutation that results in specific loss of ribosomal ribonucleic acid (RNA) synthesis and some reduction in messenger RNA synthesis. When the strain was grown in glucose medium at a restrictive temperature, RNA accumulation ceased, but both messenger RNA and protein synthesis continued for an extended time. Because carbon metabolism was slowed drastically when strain AA-157 was placed at the restrictive temperature, this phenotype can be compared with carbon depletion conditions present during diauxic lag. However, the pheontype of mutant AA-157 differs from shift-down conditions in that guanosine-3',5'-tetraphosphate levels are unaffected; therefore, a different site is affected. This mutant strain (AA-157) thus shows many characteristics similar to an aldolase mutant previously reported (Bock and Neidhardt, 1966). However, the mutation occurred in a different position on the E. coli genetic map, and furthermore, aldolase was not temperature sensitive in strain AA-157. In this paper we present a study of macromolecular biosynthesis in this mutant.

In bacteria, the rate of ribonucleic acid (RNA) synthesis varies directly with the growth rate of the organism (22, 23, 26). The rate of RNA accumulation can be specifically varied by the medium components that determine the growth rate, e.g., carbon source, nitrogen source, etc. (21). When a bacterial strain with normal ("stringent") RNA control depletes its supply of an essential nutrient, RNA accumulation will- come to an abrupt halt (21). This phenomenon has been extensively studied in relation to amino acid supply. A mutant defective in control of RNA synthesis (relaxed), which produces RNA during amino acid starvation, was discovered and has provided much impetus for research into the regulation of RNA synthesis (4, 27). During amino acid starvation, a stringent strain will cease accumulation of RNA by a noncoordinate shutdown of the production of its stable RNA species (ribosomal RNA [rRNA], transfer RNA [tRNAJ), whereas messenger RNA (mRNA) will continue at a similar or slightly reduced rate (14, 19, 25). The molecular basis for this control seems related to the tRNA binding sites on the ribosome (7, 11, 13). 'Journal paper no. J-7498 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project

no. 1747.

'Present address: Division of Biological Sciences, California Institute of Technology, Pasadena, Calif. 91109. 92

Similarly, RNA accumulation ceases after the depletion of a carbon source. This phenomenon has been most frequently studied during diauxic growth (21). RNA accumulation is again controlled by a noncoordinate production of RNA species (9, 10, 14); however, this control seems independent of the amino acid control system (28). We have isolated a temperature-sensitive (ts) mutant of Escherichia coli with a defect in RNA synthesis at the restrictive temperature. This organism shows many characteristics similar to those of an aldolase mutant previously reported (3, 4); however, the mutation lies in a different position on the genetic map and aldolase is not temperature sensitive in this strain. When grown in glucose medium at a restrictive growth temperature, RNA accumulation abruptly ceases. Because carbon metabolism is slowed drastically when this organism is placed at the restrictive temperature, temperature inactivation of RNA synthesis can be compared with the carbon depletion conditions present during a diauxic lag. In this paper we present a study of macromolecular biosynthesis in this mutant. MATERIALS AND METHODS Bacterial strains. Strains of E. coli used in this study were: NF-58, a strain of K-12 auxotrophic for arginine (argA) and methionine (metB) that exhibits stringent control of RNA synthesis by amino acids

924

FREY, NEWLIN, AND ATHERLY

(rel+); NF-59, a strain derived from NF-58 (8) exhibits relaxed control (rel) of RNA synthesis; AA-157, a-ts mutant derived from NF-59 that is auxotrophic for arginine, methionine, leucine, valine, isoleucine, and thiamine, exhibits relaxed control of RNA synthesis by amino acids (rel), and is streptomycin resistant (str); AA-156, an isogenic derivative of strain AA-157 with which it differs only near the rel allele, which is stringent for RNA control in strain AA-156; and strain KL16, an Hfr that is streptomycin sensitive (str+) and in which chromosome transfer begins near argA (54 min) on the E. coli linkage map. Growth conditions. Chemically defined media were made by supplementing a minimal salt buffer (F-buffer) consisting of (grams per liter): Na2HPO4, 5.8; KH2PO4, 3.0; NaCl, 0.5; NH4Cl, 1.0; and MgSO4, 0.12. For minimal medium, F-buffer was supplemented with 40 jig of L-arginine, L-leucine, and L-methionine per ml and with thiamine (1 /Ag/ml) and a carbon source at the following concentrations: glucose and other hexoses, and pentoses, 0.5%; glycerol, a-glycerophosphate, and lactate, 1.0%. An enriched medium was prepared by supplementing the minimal medium with the remaining 14 L-amino acids (40 4g/ml). A broth medium containing 1% tryptone, 0.5% yeast extract, 0.5% NaCl, and 0.1% glucose was also used (1). Liquid cultures were grown aerobically on a rotaryaction shaker at 30 or 43 C. Growth was measured turbidometrically in a Beckman DU-2 spectrophotometer at 350 nm. Both colorimetric and isotopic methods were used to assay synthesis of macromolecules. These procedures have been fully described (1). Isolation of mutant. A procedure was devised to isolate ts mutants that do not make RNA at the nonpermissive growth temperature (43 C). The parental strain (NF-59) was grown in broth, centrifuged, and resuspended in buffer (pH 6.0) with 50 Mug of N-methyl-N'-nitro-N-nitrosoguanidine per ml (Aldrich Chemical Co., Milwaukee, Wis). After 30 min of incubation at room temperature, the cells were centrifuged, washed to remove the mutagen, resuspended in enriched medium, and grown for several hours at 30 C to allow segregation of the mutants. ts mutants defective in RNA production were selected by the rationale that rel strains that did not produce RNA at 43 C would not incorporate a significant quantity of exogenous uridine. The culture was centrifuged and resuspended in enriched medium lacking arginine (to create starvation conditions) and after 10 min at 43 C was given a quantity of highspecific-activity [3H]uridine (1.75 ml of culture was combined with 0.07 ml [1.5 ,ug] of [3H]uridine [22 Ci/mmol]). Non-mutants and ts mutants unrelated to RNA synthesis incorporate large quantities of uridine during the 3-h incubation. After incubation the cells were chilled in ice, washed, and resuspended in minimal medium containing 15% glycerol. The cells were frozen at -70 C and samples were taken at intervals to determine the viability. Wild-type and unrelated mutants die during storage due to 3H decay promoted by the large quantity of label incorporated, but RNA-defective mutants remain viable. Indeed,

J . BACTrERIOL .

the viability in the [3Hjuridine-treated culture decreased from 0.4 x 108 to 0.4 x 104 cells/ml in 20 days, whereas the viability of a control culture decreased only from 0.7 x 108 to 0.15 x 108 cells/ml. After 20 days of storage, samples of the cultures were plated on broth-agar plates and grown at 30 C. ts mutants were detected by replica plating onto additional plates, incubating at 43 C, and selecting nongrowers. From a large number of mutants, strain AA-157 was selected for careful study. Enzyme assays. Cultures of cells were grown on Penassay broth (Difco), harvested by centrifugation during exponential growth (4 x 108 cells/ml), washed twice with F-buffer, and resuspended in 2 volumes of 0.05 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7.8). The cells were disrupted by sonic oscillation with a Biosonic II (Bronwill Scientific Corp., Rochester, N.Y.), using a microprobe set at an intensity of 50. Sonic treatment was carried out for 1 min per ml of cell suspension. Debris was removed by centrifugation at 17,000 x g for 20 min, and the supernatant fluid was dialyzed overnight against Tris buffer (1,000 volumes). Aldolase activity was determined by a coupled assay previously described (3). The reaction mixture consisted of 3 x 10-' M nicotinamide adenine dinucleotide; 1.67 x 10-2 M fructose-1,6-diphosphate (pH 7.4); 2.67 x 10-2 M glycine; 1.73 x 10-2 M Na2HAsO4 7H20; 6 units of glyceraldehyde-3-phosphate dehydrogenase in 3 M NH4S04 (Sigma Chemical Co.); and 100 to 300 Mg of crude protein extract in 1 ml. Assays were carried out in 1-ml quartz curvettes. The increase in absorbance at 340 nm was measured at 30 C in a Beckman DU-2 spectrophotometer and at 43 C in a Beckman Acta II recording spectrophotometer with a temperature-regulated cell holder. All assays were performed in the region where activity is proportional to the amount of protein added. Protein was measured spectrophotometrically by the method of Lowry et al. (16). Extraction of DNA and RNA and hybridization. Deoxyribonucleic acid (DNA) was extracted from cultures of strain NF-58 by the method of Miura (18). The DNA was subjected to two phenol (pH 9) extractions and an intermediate treatment with ribonuclease I, as described. The DNA was then further deproteinized three times with chloroform-isoamyl alcohol and precipitated twice with isopropanol. DNA so prepared was considered free of RNA and protein, and generally a spectral ratio (260/280) greater than 1.95 was achieved. rRNA was extracted from washed ribosomes of strain NF-58. Washed cells were broken by grinding in alumina. Deoxyribonuclease (10 4g/ml) and 0.01 M Tris (pH 7.0)-0.01 M MgCl2 buffer were added, and the debris was centrifuged off at 10,000 x g for 15 min and then at 12,000 x g for 30 min. The supernatant liquid was then centrifuged at 45,000 rpm for 60 min in a Beckman no. 50 rotor, and the resulting ribosome pellet was resuspended in buffer (0.01 M Mg, 0.01 M Tris). The RNA was then extracted by the hot phenol method of Salser et al. (24). The purified rRNA was frozen in 0.1 x SSC (0.15 M NaCl, 0.015 M sodium citrate) at -20 C until use.

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ts MUTANT OF E. COLI

For preparation of labeled RNA, cells were grown in arginine and containing 200 Ag of streptomycin per enriched media (1). During balanced growth of the ml. These plates were incubated at 30 C for 24 to 36 h. culture, [9H]uridine was added (1 uCi/gg) and incu- To detect recombinants that were not temperature bated for 1 min, and incorporation was stopped sensitive, samples of the mating mixture were plated rapidly by the addition of an alcohol-acetate-phenol on broth agar containing 200 Mg of streptomycin per solution (17). RNA was extracted, yielding 98% recov- ml and incubated overnight at 43 C. To obtain a recombinant that was temperature sensitive and rel+, ery. This RNA was hybridized to DNA loaded on 22-mm membrane filters after denaturation in 0.20 N conjugants of strains X-435 and AA-157 were spread NaOH. Radioactive RNA (0.10 to 0.25 ,g at 10,000 to on enriched agar lacking arginine and containing 200 15,000 counts/min per jg) was added to 0.50 ml of 2x Mg of streptomycin per ml. The plates were incubated SSC in the presence of DNA-loaded filters with and at 30 for 36 h, replica plated onto the same, and grown without 10 ug of rRNA. The mixture was incubated at 42 C. Temperature-sensitive colonies were picked for 20 h at 66 C in a sealed vial, washed on both sides from the 30 C plates and tested for rel+. One was with 50 ml of 2x SSC, and incubated for 1 h at 25 C in selected and called strain AA-156, and was used for 2.0 ml of 2x SSC containing 40 Mg of deoxyribonu- further study. clease-free ribonuclease per ml. Both sides of the filters were again washed with 50 ml of 2x SSC, the RESULTS filters were dried, and radioactivity was determined. Seventy to eighty per cent of the input [3H]RNA Strain AA-157 was isolated from E. coli strain formed hybrids in uncompeted controls at RNA/DNA NF-59 as a mutant defective in RNA biosyntheratios of 1:1,000. For more details of the hybridization sis. When strain AA-157, growing in glucose procedure see Kennell (12), from which this procedure minimal medium at 30 C, was transferred to was derived. Estimation of ppGpp levels. The presence of 43 C, RNA and protein accumulation ceased guanosine-3',5'-tetraphosphate (ppGpp) in cell cul- (Fig. 1B). In contrast, in a glucose-enriched tures was determined by the method of Cashel (5); to medium, RNA accumulation similarly ceased, 1 ml of cells, 50 MCi of [32PJorthophosphate (NaH,PO4 but protein synthesis proceeded at the 30 C rate for up to 3 h (Fig. 1A). The significance of this was at a final concentration of 2 x 10-4 M) was added, and samples (100 ul) were removed at various times observation will be discussed later in the paper. and combined with an equal volume of 2 N formic It became apparent that strain AA-157 was a acid. The remainder of the procedure was followed metabolic mutant when it was discovered that exactly as described (5). growth and macromolecular synthesis did not Genetic cross. A cross was performed between cease at 43 C when glycerol was the sole carbon strain KL-16 (Hfr, str arg A+) or X-435 (Hfr, str, rel+ argA+) and the temperature-sensitive strain AA-157 source in the medium. A detailed study re(F-, str argA rel-). The procedure has been fully vealed that strain AA-157 displayed its ts described (1). To detect recombinants argA+ str, phenotype when grown on a variety of six- and samples were plated on enriched agar (1) lacking five-carbon sugars, but growth was not impeded

x

'D

l0

0

x

4)5

010

c

9

4

.

c

L

:3~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

8

30

9 0 3 630120 60

'90

120

30

6.0

-0

Time (min)

FIG. 1. RNA and protein synthesis in strain AA-157. Cells growing in glucose-enriched (A) or glucose minimal (B) medium were labeled with [3Hluridine (2.5 mCi/mmol, 0.08 gmol/ml) and ['4C]phenylalanine (0.16 mCi/mmol, 0.125 Mmol/ml). After 2 h of incubation in the presence of label, samples of the culture were placed at appropriate temperatures and samples taken at the time intervals shown. Symbols: (0) RNA synthesis at 30 C; (A) protein synthesis at 30 C; (0) RNA synthesis as 43 C; (A) protein synthesis at 43 C.

on a number of two- and three-carbon compounds (Table 1). Growth also was arrested in glycerol medium at 43 C if minute quantities of glucose (10 ,ug/ml) were added; however, when a-glycerophosphate was used as an energy source, added glucose did not inhibit growth at 43 C. These results are very similar to found by Bock and Neidhardt (3) in an E. coli strain temperature sensitive for fructose-1, 6-diphosphate aldolase. A stringent derivative of strain AA-157 was prepared (see Materials and Methods) and was found to grow similarly on the carbon sources tested (Table 1, strain AA-156). In contrast to growth in liquid medium, both strains AA- 156 and AA- 157 formed only minute colonies on glycerol-agar plates after 6 days of incubation at 43 C. Apparently, the mutant strain cannot undergo continued division before eventually stopping. These phenotypic characteristics were similar to those of a ts mutant reported by B6ck and Neidhardt (2, 3). Similar to their mutant, strain AA-157 was found to have a markedly reduced level of fructose-1,6-diphosphate aldolase activity when grown at 30 C and an even lower level when grown at 43 C (Table 2). The level of aldolase activity found in crude extracts of the mutant strain AA-157, however, was reduced to only 10% of its parent when assayed at 30 or 42 C. In contrast, Bock and Heidhardt (2) observed a much greater reduction in the aldolase levels in their mutant (less than 1% of its parent). In addition to these findings, several other parameters of strain AA-157 were measured, i.e., radiorespirometary study of glucose

at 43 C

TABLE 1. Doubling times

on

various carbon

sources

Doubling time Strain

AA-157 (rel)

AA-156 (rel+)

NF-59

(min)

Compound

30C

43C

120 130 130 135 135 125 135 100 170

_a

130 135

90 120

Gluconate

120 125 120 120

Glucose Glycerol Glucose-6-phosphate

55

34

105 57

63 35

Glucose Glucose-6-phosphate Fructose-6-phosphate Glycerol a-Glycerol-phosphate Galactose Mannose Gluconate Lactate

a

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FREY, NEWLIN, AND ATHERLY

926

Glucose Glycerol a-Glycerol-phosphate

Values could not be obtained owing

90

to lack of growth.

TABLE 2. Fructose-1,6-diphosphate aldolase activity Strain

NF-59 AA-157 AA-157R1b AA-157R2 AA-157REc

Growth temp (C)

No. of expts

30 43 30 43 30 30 30

Enzyme activitya 30 C

42 C

3 2 4 2 1 1

7.0 7.0 0.70 0.20 1.13

3.5

1

7.4

0.64

0.90 4.2

aEnzyme assays were done at 30 or 42 C as described in text. Activity is the number of micromoles of fructose-1,6-diphosphate cleaved per hour per milligram of protein as measured by the number of micromoles of nicotinamide adenine dinucleotide reduced per hour per milligram of protein. b R refers to revertant. c RE refers to recombinant strain.

metabolism and the fate of Cl- and C6-labeled glucose; all characteristics measured were identical in strain AA-157 to those described by Bock and Neidhardt (3) in their fructose-1,6diphosphate aldolase mutant. Studies on the residual aldolase activity in strain AA-157 demonstrated that it was no more temperature sensitive than aldolase from the parent strain. Aldolase activity was partially purified and subjected to temperature inactivation, yielding identical inactivation curves for mutant and parent aldolase (Fig. 2). Thus, the aldolase enzyme in mutant strain AA-157 appears not to be temperature sensitive. On the other hand, the synthesis of aldolase or the regulation of synthesis could still be temperature sensitive. Genetic studies. Strain AA-157 was found to have a reversion frequency of 1.5 x 10-1 (measured on both minimal medium and broth agar plates) and was thus assumed to be a point mutation. Several revertants tested for aldolase activity showed slightly increased levels of aldolase activity relative to the mutant, but wild-type levels were not restored (Table 2). However, examination of the growth characteristics of several revertants showed growth rates on various carbon sources identical to those of the parent strain at 30 and 43 C. A derivative of strain AA-157 was made by interrupted mating with strain KL-16. Recombinants of these two strains were selected that were still temperature sensitive but possessed wild-type levels of aldolase (Table 2). Thus, aldolase levels can be restored to normal while still retaining the ts phenotype. The mutation in strain AA-157 was mapped by interrupted mating using Hfr strain KL-16,

VOL. 121, 1975

ts MUTANT OF E. COLI

0

50 -

C.)

25 Cw3 0 -J 0

-C

o

z C.)

lo1

o

a

o\

w

0LJ 5

2

4

MINUTES

6 8 AT 55C

10

FIG. 2. Heat inactivation phate aldolase. Aldolase wa ospartially (10fold) by precipitation betwee?n 50 and 70%oammonium sulfate at pH 7.4 and 4 C in 0.05 M Tris buffer from the 100,000 x g supernatant liquid of the cell extracts of strains NF-59 or AA-157. 'The precipitated enzyme was redissolved and dialyze?d against 0.05 M Tris. Samples of the enzyme (0.1() ml) were placed in test tubes and immersed in a 55 C water bath. Samples were removed at intervals aind cooled, and aldolase activity determined. EnzymEe activity i's expressed as a percentage of zero time. Sy mbols: (0) Strain NF-59, (O) strain AA -157.

puripied

'_ \,

3&

LIl&"t as s s-s X.

and the mutation was located at about 54 min on the E. coli map, within 1 min of the argA locus and very close to the rel locus (Fig. 3). Strain AA-157 showel a recombination frequency of 20% between argA and the ts locus after mating with Hfr strain KL-16. argA also was found to cotransduce with the ts locus at a frequency of 22%. This map position is different from that reported by Bock and Neidhardt (3) (60 min) and thus would suggest that the defect in strain AA-157 is not in the structural gene for aldolase. The possibility remains, however, that the mutation represents a defect in a gene responsible for regulation of aldolase activity. RNA synthesis. To determine whether RNA production was completely halted (as opposed to a rapid turnover of RNA) in mutant strain AA-157, the initial rates of RNA synthesis at 43 C were determined. A culture of strain AA-157 growing exponentially in minimal medium at 30 C was placed at 43 C. At time intervals after the temperature change, samples of the culture were combined with [3H ]uridine,

927

and the incorporation of the label into trichloroacetic acid-precipitable material was determined. A steep initial rise in labeling occurred, followed by a rapid decrease in rate after 5 to 10 min (Fig. 4). This suggested that RNA is being actively synthesized in strain AA-157 at 43 C but must be rapidly destroyed, since no accumulation was observed after 30 min at 43 C. Also, the rate of RNA synthesis seemed unaffected by the length of time at 43 C, since similar curves were observed after 90 min of incubation (Fig. 4). mRNA production. The previous experiment indicates that an RNA species was produced in strain AA-157 at 43 C that was rapidly degraded. Also, protein synthesis proceeded when strain AA-157 was grown at 43 C in glucoseenriched medium. Thus, it would seem likely that mRNA synthesis occurred in strain AA-157 at the restrictive temperature. Consequently, synthesis of mRNA was examined by studying the production of the protein product of a specific mRNA, A-galactosidase. If protein synthesis is normal in strain AA-157 at 43 C, then the production of 0-galactosidase is a valid measure of d-galactosidase mRNA synthesis, which in turn should reflect mRNA synthesis in general. s-Galactosidase synthesis was induced with isopropyl-f3-D-thiogalactopyranoside (IPTG; 10-i M) in cells growing in glucose-enriched medium (Fig. 5). After an initial lag, ,8-galactosidase was produced linearly with time in the

400( E U)

C

300(

Cs

C: n E 0

200(

0 a)

100(

Mi nutes FIG. 3. Interrupted mating between strains AA-157 and KL-16. Symbols: (0) argA+ recombinants; (A) ts+ recombinants.

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FREY, NEWLIN, AND ATHERLY

928

previous results indicate that, although mRNA synthesis in strain AA-157 was depressed, it was increased in relative proportion to the total X RNA produced. Thus, the production of stable RNA species must have been severely reduced, or stable RNA had become unstable. To examf m\ ine more closely the hypothesis that the majority of RNA produced in strain AA-157 at 43 C

mRNA, DNA-RNA hybridization studies were performed. Cells were grown in an enriched medium and,

6

L

was

30

L4-

AA- 157

A 2

20

M nu tes

FIG. 4. Initial rates of RNA synthesis. Strain AA-157 was grown in glucose minimal medium and transferred to 43 C, and label was added as described in the text. 3.25 uCi of [3H]uridine was added to 15-ml samples of the original culture at 0 (0), 10 (0), 30 (A), 60 (0), and 90 (-) min after the temperature change. A control, utilizing strain NF-59, was run simultaneously at 43 C and resulted in logrithmic incorporation for greater than 100 min and over 100,000 counts/min.

wild-type parent cells (NF-59) growing at 30 and 43 C and in strain AA-157 growing at 30 C. Strain AA-157 at 43 C, however, could be only minimally induced after a long lag (Fig. 5). This effect, probably because of severe catabolite repression caused by the metabolic defect in strain AA-157, was counteracted by the addition of 10-s M 3',5'-cyclic adenosine monophosphate (cAMP) to the growth medium. The presence of cAMP stimulated 13-galactosidase production in both strains NF-59 and AA-157 at 30 and 43 C. In the presence of cAMP, the rate of ,B-galactosidase production in strain AA-157 at 43 C was 1.4 times greater than its 30 C rate; however, f3-galactosidase production in strain AA-157 at 43 C was only 25%c of the rate of strain NF-59 at 43 C. These results indicate that mRNA production does indeed occur in strain AA-157 at 43 C. Although the rate was stimulated over its 30 C rate, it was depressed in comparison with strain NF-59. No change in total protein or RNA synthesis over the normal was detected when cells were grown in the presence of cAMP.

Relative amounts of RNA produced. The

10

/ /I

-

/

-

C

/

/

A

1

B

B

>

/

NF59

l /

50

_ / If

d , 15 20 25 T ime (m n) FIG. 5. Induction of ,B-galactosidase in strain AA-157 and NF-59. Exponentially growing cells in glucose-enriched medium were induced for j-galactosidase production by the addition of IPTG (10-3 M) 0

5

10

with or without cAMP (10- M). At the time intervals shown, duplicate samples were withdrawn and assayed described theC;text. (0)C;Cells inducedas with IPTG in at 30 (0) Symbols: IPTG at 43 (A)

IPTG and cAMP at 30 C; (A) IPTG and cAMP at 43 C. One activity unit was defined as the change of 0.001 optical density unit at 420 nm per min per 3 x 108 cells.

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ts MUTANT OF E. COLI

after 15 min of incubation at 43 C, were given a 1-min pulse of [3H Juridine. To prevent the anticipated rapid decay of RNA, growth was stopped by the addition of an alcohol-acetatephenol solution to the medium (17). This mixture stops the reaction instantaneously without physically altering the structure of the RNA. RNA was then extracted and hybridized to E. coli DNA on membrane filters as described in Materials and Methods. By using the criterion established by Kennell (12) for determining the type of RNA, strain AA-157 was found to produce a much reduced amount of stable RNA species (Table 3). Approximately 10 to 15% of the RNA produced at 43 C in strain AA-157 was stable (rRNA and tRNA), whereas 40 to 50% in strain NF-59 was stable RNA, as determined by this technique. These data suggest that the majority of RNA produced in strain AA-156 at 43 C was messenger and thus explain the lack of accumulation of isotopically labeled RNA at 43 C (Fig. 1). Protein synthesis. An interesting aspect of strain AA-157 is its growth characteristics in minimal medium. Whereas in enriched medium protein is produced at a definite rate, in minimal medium no protein accumulates. The metabolic blockage caused by the aldolase enzyme at 43 C would lead to a shortage of metabolic intermediates that, in turn, would cause a

929

shortage of precursors for amino acid biosynthe-

and thus result in a modified amino acid starvation. To test this hypothesis, strain AA-157 was grown in minimal medium and transferred to 43 C. After 30 min of incubation, a portion of this culture was given a complete supplement of amino acids. The amino acid supplementation definitely stimulated the rate of protein synthesis at 43 C in strain AA-157 (Fig. 6), and thus growth in minimal medium seemingly limited protein synthesis by amino acid starvation. A similar observation was made when a specific protein was studied. No ,-galactosidase could be induced in strain AA-157 growing on minimal medium at 43 C even in the presence of cAMP. When an amino acid supplement was given an induced culture of strain AA-157 on minimal medium, ,8-galactosidase was produced after a short (3.5-min) lag (Fig. 7). To prove that the amino acid supplement corrected protein synthesis by supplying amino acids and did not simply alleviate a severe form of catabolite repression known to occur when cells are starved of both amino acids and carbon sources (20), IPTG and cAMP were filtered from the medium before the amino acids were added. sis

j%-Galactosidase was produced at a similar, though decreasing rate when compared with the situation when inducer and cAMP

were

left in

TABLE 3. Hybridization of pulse-labeled RNA to DNA trapped on a filtera Part

Strain

Counts at RNA/DNA ratio of:

DNA (ug/filter)

Input A

B

NF-59 AA-157

200 100 100 150 200

Strain

DNA (ug/filter)

NF-59

150 150 200

AA-157

150 200

_

Hybridized

Input

Hybridized

13,354 4,743 8,339 13,907 23,569

942 250 282 792 792

707 190 209 494 538

33,780 12,483 14,096 28,500 38,060

mRNA (%) 53 50 80 87 89

Counts hybridized

Input counts 706 706 942 792 1056

lig

mRNA

-RNA

+rRNA

515 515 707 538 553

340 300 355 462 484

66 58 51 83 88

a The amount of DNA on filters was always kept at 100 to 200 even at low ratios to prevent a concentration effect which would decrease the efficiency of hybridization. Part A: RNA pulse-labeled with [3HJuridine was hybridized over a wide range of RNA/DNA ratios. Reported here are the values obtained at 1:1,000 ratio, at which all RNA species hybridize, and 1:20 ratio, at which only mRNA hybridizes. The percentage hybridized at a 1:1,000 ratio of RNA/DNA was set at 100%, and the relative percentage hybridized at 1:20 RNA/DNA is the percentage of mRNA. Part B: RNA was hybridized to DNA (1:1,000) in the presence and absence of a 40-fold (10 ug) excess of unlabeled rRNA. The percentage of the counts remaining hybridized in the presence of rRNA is the percentage of mRNA in the sample.

930

FREY, NEWLIN, AND ATHERLY

J . BACTrERIOL .

levels of aldolase and are still temperature sensitive (Table 2). Reversion frequencies of mutant strain AA-157 suggest that it is a single / mutation. Aldolase activity in revertants, how,I' E 3.5 ever, is not present at wild-type levels, indicat,/' ing that the mutation may be a complex one within a single gene or conceivably a regulator X _ gene for aldolase activity. Thus, genetic as well o~~~~~ as biochemical data indicate that the mutation _is not in the aldolase structural gene. Other _. enzymes assayed for activity include phosphofructokinase, triosephosphateisomerase, and glycerol-3-phosphate dehydrogenase, and all were present at wild-type levels. No known mutation in carbohydrate metabolism occurs at minute 54 of the E. coli genetic map, where the 15 30 75 mutation in AA-157 is located. 90 Minutes The genetic lesion causes a cessation of RNA FIG. 6. Protein synthesis in minimal and supple- accumulation at 43 C, which is most probably a mented minimal medium. Strain AA-15,7 cells grow- secondary phenotypic effect of the mutation. ing in glucose minimal medium were preilabeled with The primary effect is a drastic reduction in the [14Clphenylalanine. At 30 min after theey had been rate of metabolism, and thus the conditions in placed at 43 C, half of the culture was supplemented strain AA-157 at 43 C are not unlike those in a with all amino acids (20 ug/ml). Symbo Is: (0) Glu- bacterium undergoing a carbon source depletion A

a~~~~~~~~~~~~ u~~~~~~~~~~~~

C

u3.0

2 .5

45

cose

60

minimal medium; (A) with amino acid supple-

ment.

15

the medium (Fig. 7). Thus ,-gallactosidase mRNA was produced in minimal rmedium at 43 C and could not be translated because of amino acid deprivation caused by the metabolic (I)l block in strain AA-157. Synthesis of ppGpp. An unusual compound -10 has been isolated from stringent bactieria under- D going amino acid starvation. The compound, >'O ppGpp, also has been detected in cells where -, stable RNA production has ceasedI during a > diauxic lag. ppGpp has been propo)sed as the -+-l molecular regulator of RNA synthesiis (6). Con- < 5 sequently, it was of interest to determine whether ppGpp accumulated in strekin AA-157 rel or strain AA-156 rel+ at 43 C . In three separate experiments, no significant accumulation of ppGpp was found in either strrain during growth or during cessation of RNA s,ynthesis at -4 the restrictive temperature. Howevi'er, normal °0 er inosrmaln 10 Iv 20 30 40 50 60 ppGpp accumulation was observed in strain T i me (m n ) AA-156 rel+ when starved for a requ ired amino FIG. 7. Stimulation of ,8-galactosidase synthesis by acid at either 30 or 42 C. amino acid supplement of minimal medium. A cul-

DISCUSSION Mutant strain AA-157 exhibits ma ny characteristics similar to the ts aldolase rmutant reHowever,, ported by Bick and Neidhardt (2, 3) Howevr a comparison between the heat s of aldolase from the mutant and its par ent showed no differences (Fig. 2). Also, re4combinant strains were constructed that possesk s wild-type

etabilty

ture of strain AA-157 growing (30 C) on glucose minimal medium was transferred to 43 C and incubated 30 min before IPTG and cAMP were added. Fifteen minutes after induction (1), samples of the cells were given an amino acid supplement in the presence and absence of inducer. Samples were withdrawn and assayed as described in the text. Symbols:

(a) Glucose minimal medium; (A) amino acid supplement; (0) amino acid supplement, IPTG and cAMP filtered out. For other details see Fig. 5.

VOL. 121, 1975

ts MUTANT OF E. COLI

as during a diauxic lag. RNA synthesis in strain AA-157 is by a noncoordinate cessation of the production of stable RNA species, whereas RNA continues to be synthesized at a reduced rate. This is indicated by the near-normal production of the specific mRNA for ,-galactosidase in the absence of RNA accumulation (Fig. 5) and the high percentage of pulse label found in RNA that is not competed out by added rRNA during RNA-DNA hybridization studies (Table 3). This is similar to the control mechanism used during a diauxic lag. Thus, the cessation of RNA production at 43 C in strain AA-157 is probably due to normal mechanisms of RNA control that E. coli utilizes during a carbon source depletion. Cessation of RNA synthesis in stringent bacteria can be elicited by the exhaustion of one of several medium components (carbon source or required amino acid supply). The noncoordinate shutdown of RNA synthesis is the typical control mechanism in all these cases. However, controls of RNA production by carbon source and amino acid supply are apparently independent of each other. The relaxed mutation has no effect on RNA synthesis during carbon source depletion even when a relaxed auxotroph is specifically starved for an essential amino acid during carbon source depletion (9). The molecular control, however, may be similar. Chloramphenicol stimulates RNA synthesis although it turns off protein synthesis. This effect is elicited through the presence of charged tRNA on the ribosome (11). Chloramphenicol also stimulated RNA synthesis during carbon depletion, an effect that would indicate a similar type of control specifically in AA-157; amino acids are needed for stimulation of RNA synthesis by chloramphenicol (data not shown). Possibly a molecular regulator for RNA biosynthesis exists that responds to specific medium components; however, these controls override each other so that depletion of one component determines the fate of RNA synthesis regardless of the supply of other components. Protein synthesis offers a central metabolic event of growing bacteria and a prime candidate for regulation of RNA synthesis. ppGpp has been implicated in the regulation of stable RNA synthesis in bacteria (6). In a rel+ strain (but not in a rel strain) of E. coli, ppGpp accumulation occurs rapidly during the shut-off of rRNA synthesis under amino acid starvation conditions. On the other hand, relaxed (rel) cells do accumulate ppGpp during step-down transitions that preferentially restrict rRNA synthesis (14, 28). When strains AA-157 or AA-156 are transferred to nonpermissive growth

931

conditions, which is similar to a step-down transition, no accumulation of ppGpp is observed. Consequently, these data suggest that ppGpp is not involved in the shutdown of stable RNA synthesis in strains AA-157 or AA-156 and thus is different from a carbon depletion elicited by a step-down transition. Thus, the possibility exists that ppGpp is not involved in rRNA tumoff during step-down conditions since turnoff can occur in the absence of ppGpp production. Conceivably rRNA synthesis may require a higher level of some necessary component (a nucleoside triphosphate?) than mRNA synthesis. ACKNOWLEDGMENTS This work was supported in part by National Science Foundation grants GB-8722 and GB-37634 and Damon Runyon Fund grant DRG-1197 to A.G.A. LITERATURE CITED 1. Atherly, A., and M. Suchanek. 1971. An unusual phenotype associated with phenylalanyl transfer ribonucleic acid synthetase mutant. J. Bacteriol. 108:627-638. 2. Bock, A., and F. C. Ntidhardt. 1966. Isolation of a

mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase activity. J. Bacteriol. 92:464-469. 3. Bock, A., and F. C. Neidhardt. 1966. Properties of a mutant of Escherichia coli with a temperature-sensitive fructose- 1,6-diphosphate aldolase. J. Bacteriol. 92:470-476. 4. Borek, E., A. Ryan, and J. Rockenbach. 1955. Nucleic acid metabolism in relation to the lysogenic phenomenon. J. Bacteriol. 69:460-467. 5. Cashel, M. 1969. The control of RNA synthesis in Escherichia coli. J. Biol. Chem. 244:3133-3141. 6. Cashel, M. 1970. Inhibition of RNA polymerase by ppGpp, a nucleotide accumulated during stringent response to amino acid starvation in E. coli. Cold Spring Harbor Symp. Quant. Biol. 35:407-414. 7. Eidlic, L., and F. Neidhardt, 1965. Protein and nucleic acid synthesis in two mutants of Escherichia coli with temperature-sensitive aminoacyl ribonucleic acid synthetases. J. Bacteriol. 89:706-711. 8. Fiil, N. 1969. A functional analysis of the rel gene in Escherichia coli. J. Mol. Biol. 45:195-203. 9. Jacobson, L. A. 1970. Regulation of ribonucleic acid synthesis in Escherichia coli during diauxie lag: accumulation of heterogeneous ribonucleic acid. J. Bacteriol. 102:740-746. 10. Jacobson, L. A. 1972. Control of stable ribonucleic acid chain initiation in Escherichia coli during diauxie lag. J. Bacteriol. 109:678-685. 11. Kaplan, S., A. G. Atherly, and A. Barrett. 1973. Synthesis of stable RNA in stringent Escherichia coli cells in the absence of charged tRNA. Proc. Nat. Acad. Sci. U.S.A. 70:689-692. 12. Kennell, D. 1968. Titration of gene sties on DNA by DNA-RNA hybridization. II. The Escherichia coli chromosome. J. Mol. Biol. 34:85-103. 13. Kurland, C., and 0. MaalOe. 1962. Regulation of ribosomal and transfer RNA synthesis. J. Mol. Biol. 4:193-210. 14. Lazzarini, R., and R. Winslow. 1970. The regulation of RNA synthesis during growth rate transitions and amino acid deprivation in E. coli. Cold Spring Harbor Symp. Quant. Biol. 35:383-390.

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coli. J. Biol. Chem. 246:420-429. 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275. Manor, H., D. Goodman, and G. S. Stent. 1969. RNA chain growth rates in Escherichia coli. J. Mol. Biol. 39:1-29. Miura, K. 1967. Preparation of bacterial DNA by the phenol-pH9-RNase method, p. 543-544. In S. P. Colowick and N. 0. Kaplan (ed.), Methods in enzymology, vol. 12. Academic Press Inc., New York. Morris, D., and Kjeldgaard. 1968. Evidence for noncoordinate regulation of RNA synthesis in stringent strains of E. coli. J. Mol. Biol. 31:145-148. Nakada, D., and B. Magasanik. 1964. The roles of inducer and catabolite represser in the synthesis of , galactosidase in E. coli. J. Mol. Biol. 8:105-127. Neidhardt, F., and B. Magasanik. 1960. Studies on the role of RNA in the growth of bacteria. Biochim. Biophys. Acta 42:99-106.

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