Magnesium Starvation of Aerobacter aerogenes · Magnesium Starvation of Aerobacter aerogenes II. Rates of Nucleic Acid Synthesis andMethodsfor Their Measurement DAVID KENNELLAND ANGELIKI

JOURNAL OF BACTERIOLOGY, Jan., 1967, p. 345-356Copyright @ 1967 American Society for Microbiology

Vol. 93, No. 1Printed in U.S.A.

Magnesium Starvation of Aerobacter aerogenesII. Rates of Nucleic Acid Synthesis and Methods for Their Measurement

DAVID KENNELL AND ANGELIKI KOTOULASDepartment of Microbiology, Washington University School of Medicine,

St. Louis, Missouri

Received for publication 26 August 1966

ABSTRACTThe rates of synthesis of Aerobacter aerogenes nucleic acids were estimated dur-

ing incubation of the bacteria in a Mg++-free medium. Deoxyribonucleic acid(DNA) synthesized during Mg++ starvation, or in the preceding exponential growth,remained acid-precipitable for 2.5 hr before breaking down to acid-soluble productsduring a period of many hours. Rates of DNA synthesis were calculated by cor-recting the net amounts of DNA per milliliter to values that would have appearedhad there been no decay. After the first few hours, this rate was constant, theamount of DNA present at the start of Mg++ starvation being synthesized every130 min. Rates of synthesis of total ribonucleic acid (RNA) were established intwo ways: (i) by measurements of the incorporation of exogeneous uracil andglucose carbon into RNA, and (ii) by the accumulation of transfer RNA (tRNA),since this component is stable during Mg++ starvation. After the first few hours,this rate was constant, the amount of RNA present at the start of Mg++ starvationbeing synthesized about every 120 min. Fractionation by gradient centrifugationrevealed that at all times of starvation the ratio of newly synthesized tRNA-rRNAwas the same as it was during exponential growth. Furthermore, newly synthesizedribosomal RNA (rRNA) became a part of polysomal structures. Thus, in theabsence of Mg++, DNA, tRNA, and rRNA were synthesized in the same relativeproportions as during exponential growth, at rates close to one-half the instan-taneous rates of synthesis in the bacteria growing exponentially at the start ofstarvation.

One way a cell or an organism could respondto an adverse environment would be to discon-tinue its synthetic activities, as occurs duringsporulation; thus, it would become less depend-ent upon its surroundings for survival. Amongmost bacteria, however, this appears to be theexception rather than the rule. Instead, duringstarvation or in the presence of specific inhibitors,the synthesis of various cell components maycontinue at different relative rates, with the pro-duction of highly anomalous protoplasm whichsometimes leads to death of the organism. Forexample, it is possible to block protein synthesiswithout inhibiting nucleic acid synthesis; this canresult in an accumulation of nucleic acids plusprotein-deficient ribosomes, e.g., in K+ starva-tion (3) and in chloramphenicol treatment (12).Whether favorable or not for survival, such re-sponse has made possible the diverse forms oflife and metabolism which now exist. Only byactively meeting the challenge were opportunitiespresented for the selective evolution of better-adapted forms.

In spite of the important role of Mg++ incellular metabolism (10), Aerobacter aerogenescontinues to synthesize its major nucleic acid andprotein components during Mg++ starvation atrates close to one-half the instantaneous rates inthe same cells growing exponentially at the onsetof the starvation. Because the ribosome contentdoes not increase during this time, the macro-molecular composition of the culture becomesincreasingly abnormal with time of starvation (5).

In this paper, the rates of synthesis of deoxy-ribonucleuc acid (DNA) and transfer and ribo-somal ribonucleic acid (tRNA and rRNA) wereestimated during Mg++ starvation. The measure-ments were complicated by the fact that theamount of exogenous precursor incorporated intoRNA or DNA per unit of nucleic acid synthe-sized changed as a function of starvation time.These changes presumably reflect specific changesin permeability or in repression or inhibition ofcertain biosynthetic reactions in the cell. Whereasthe method used for estimating DNA synthesis isrelatively straightforward, it has been necessary

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KENNELL AND KOTOULAS

to analyze in some detail the problem of measur-ing RNA synthesis in a system, such as this, inwhich rRNA is breaking down as rapidly as it isbeing formed (see Discussion).

MATERIALS AND METHODS

General procedures. The organism (A. aerogenesstrain 1033), the conditions of culture for exponentialgrowth and for starvation, the preparation of extracts,the purification of RNA, centrifugation through gra-dients of sucrose and corrections for losses were de-scribed in the preceding paper (5). A. aerogenes strainU3 (uracil-requiring) was selected from strain 1033by the penicillin technique as modified by Gorini andKaufman (4). The estimations of acid-insoluble radio-activity and the preparation of extracts containingpolysome structures were described in the precedingpaper (5). The procedures used to purify RNA carbon,free from all other bacterial carbon, are included in anAppendix to this paper.

Determination ofbase composition of total RNA. Toestimate the relative contributions of carbon fromexogenous uracil and glucose to RNA pyrimidines(Table 1), it is necessary to know the base compositionof the bacterial RNA. This was determined by growingcells for several generations in the presence of inor-ganic P3a before purification of RNA and separationof its labeled mononucleotides by electrophoresis asdescribed in the Appendix to this paper. The valuesobtained were: C = 22.7%; A = 24.4%; G = 32.8%;U = 20.0%. These values agree closely with thosepreviously determined for A. aerogenes RNA (1).

Relative rates of synthesis. Rates of synthesis werecompared to the rates extant at the time Mg++ wasremoved from the medium. In an exponentially grow-ing population, the rate of increase of any compo-nent, e.g., total RNA, can be expressed by either (i)its instantaneous rate of formation or (ii) the ex-ponential rate of growth. Thus, in exponential growth:R = Roekt, where k is the growth constant and equalsIn2/T. The rate of increase is: dR/dt = Ro(ln2/T),

e(ln2/T)t. The instantaneous or tangential rate at atime t = 0 is (dR/dt)o = Ro(ln2/T). [R = RNA permilliliter at time t. Ro = RNA per milliliter at time 0.T = exponential mass doubling time (38 min in ourpopulations).] Thus, (dR/dt)o = 0.0183 units per minper unit ofRNA, or 55 min to synthesize an Ro amountof RNA.

This figure reflects the biosynthetic activity of ex-ponentially growing cells at any instant, and is thefigure to contrast to biosynthetic rates during lineargrowth periods, such as occur during Mg++ starvation.For example, if, after exponential growth, bacteriagrow linearly at such a rate that the amount per milli-liter of any component present at the onset of lineargrowth is synthesized every 55 min, this means thatthose elements of the cell (or more likely the one stepwhich has become rate-limiting) which determine thebiosynthetic rates are operating at the same rate asthey did during exponential growth-the only differ-ence being that they are not being reproduced ex-ponentially.To contrast biosynthetic rates in this manner, rates

of synthesis, unless specified otherwise, refer to ratesper milliliter of culture. The instantaneous rate ofsynthesis in exponentially growing cells specificallyrefers to that synthesis in 1 ml of culture at the initia-tion ofMg+ starvation, i.e., containing the same massof cells per milliliter as are present in the Mg++-starved culture at the start of starvation. The normal-ization procedures were described previously (5).

Radioactive compounds. The following compoundswere products ofNew England Nuclear Corp., Boston,Mass.: uracil-2-CH4 (0.1 mc/0.38 mg); uracil-5,6-H3(5 mc/0.4 mg); glucose-Cl4, uniformly labeled (0.50mc/0.475 mg); methyl-thymidine-H3 (14 c/mmole);and thymidine-2-C'4 (0.12 mc/mg). Guanine-8-HI(378 mc/mmole) was from Nuclear-Chicago Corp.,Des Plaines, Ill.

RESULTS

RNA synthesized during magnesium starvation.To estimate the relative amount of rRNA com-pared with tRNA that was synthesized duringMg++ starvation, bacteria were exposed to uracil-C4 for 1 min and then to a large excess of un-labeled uracil for 30 min; only the stable nucleicacids contained radioactivity, and the labeledribosomal components had attained sedimenta-tion rates of the completed products but had notbegun to decay (5). The bacteria were chilled andwashed, extracts were prepared, and the RNAwas purified for sucrose gradient centrifugation.The results are unambiguous: the relative amountsof rRNA to tRNA being formed at any timewere the same (Fig. 1). The only apparent dis-crepancy, especially in the pattern at 8 hr, maybe a slightly larger amount of 16S rRNA com-pared with 23S rRNA. However, this discrepancysimply reflects a slower formation of rRNA dur-ing starvation since in exponentially growing cellsthe formation of a 23S component, which is stableto the procedures used to purify RNA, requiresmore time than does the formation of a stable16S component. If the chase was extended for 90mi, the patterns did become normal (unpub-lished data).

Formation of ribosomal structures. AlthoughrRNA was synthesized at a normal rate with re-spect to the rate of tRNA synthesis (above), itwas possible that during Mg-H starvation newlysynthesized rRNA polymers could not be used toassemble new ribosomes or new polysomes. Thispossibility was examined by treating bacteriaexactly as described above, but extracts were pre-pared so as to minimize loss of polysome struc-tures. Gradient sedimentation oP these extractsshowed that at any time during Mg++ starvationnewly synthesized RNA was being incorporatedinto 70S ribosomes, and more significantly, intopolysomes (Fig. 2). After 21 hr, the proportion ofnewly synthesized RNA that sedimented as

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MAGNESIUM STARVATION OF A. AEROGENES. II

23S 16S the time Mg++ is removed (10), so that theseA + structures must be replenished continuously dur-6000o - 15 |ling the starvation.

Absolute rates of total RNA synthesis estimatedB 1 \ by precursor incorporation. The rate of total RNA

1500- / synthesis can be estimated by the incorporationof isotope derived from a precursor molecule. It

!0M / / t\is important to recognize the limitations of this10oo- c method (see Discussion). At various intervals of

1500 o starvation as wel as during exponential growth,A incorporation of the following precursors intooL r zB RNA was measured for periods of 90 min: (i)

1000 5mm uracil-H3 into the RNA of strain U3, a uracil-0D0 requiring mutant of A. aerogenes; (ii) uniformly600

labeled glucose-C14 into RNA when glucose wasQj-500- j / 1 1 l \ / \ the sole source of carbon [the accumulation of

* .E/ T 5hr this RNA-C14 in exponentiaUy growing A. aero-150- genes 1033 is shown in Fig. 3 (see Discussion)];

300- 0L F|A (iii) purine-H3 or pyrimidine-H3 in addition to8hr uniformly labeled glucose-C14 by wild-type bac-

teria with glucose as the major carbon source[the accumulation during exponential growth ofRNA-H3 derived from guanine-IP is also shown

0-5D2 Xhr70S

0 6 12 18 24 30 36FRACTION NUMBER

FIG. 1. Sedimentation characteristics of newly syn-thesized RNA in Aerobacter aerogenes at various timesofMg++ starvation. Acid-precipitable counts per minutein fractions collected after sucrose density gradient A Icentrifugation ofpurified RNA. Bacteria (300 to 1,000 ifiml), growing exponentially (0) or starved of Mg+ forthe times indicated, were exposed to 10 uc of uracil-2-C'4 (0.1 mc/0.38 mg) for I min, then to uracil-C'2 (30 - 0to 100 mg) for 30 min before chilling. Recovered radio-activities were not normalized to correct for losses. /0 min.Centrifugation was for 7 hr at 37,000 rev/min and 4 Cin an SW39 rotor (Spinco).

polysome material was not significantly different 2 hrfrom that of exponentially growing cells, al- t/though there may have been a relative increase in 7hrthe number of 30 and 50S subunits which were"held-up" as starvation progressed (tubes 30 i 2/hrthrough 34). 21 hrThe same bacteria from which these extracts ,<

were derived had been exposed to uracil-H3 only Q'during the exponential growth phase to observe 6 2 18 2 3 36the sequence of steps in the breakdown of the FRACTION NUMBERpolysomes to acid-soluble products (Fig. 9 ofpreceding paper). This clearly demonstrated that FIG. 2. Sedimentation characteristics of newly syn-the processes of breakdown and resynthesis of thesizedribosomal structures in Aerobacter aerogenesr.bosoma oma eral,ncldin polysomes, were at various times of Mg++ starvation. Bacteria wereri'osomalmaterial,including polyso' w labeled by the procedures in Fig.I except that the chaseoccurring simultaneously in the starving cells. of C'2-uracil was for5 min. Radioactivities were notThis was not unexpected since protein synthesis normalized to correct for losses. Centrifugation was forcontinues at close to one-half the instantaneous I hr at 37,000 rev/min and 4 C in an SW 39rate of the same cells growing exponentially at rotor (Spinco).

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in Fig. 3 (see Discussion)]. For these measure-ments, it was necessary to develop a simple pro-cedure for purifying microgram amounts ofRNA carbon free from all other cell carbon (seeAppendix).The rates of synthesis estimated by the three

procedures are similar, but the last method (3) isparticularly interesting because it demonstratesthat the amount of exogenous base incorporatedinto RNA is not always proportional to theamount of RNA synthesized. A. aerogenes 1033cells, growing exponentially or at a defined timeof Mg++ starvation, were exposed simultaneouslyto uracil-H3 and glucose-C'4 (uniformly labeled);after 35, 50, 70, and 85 min, RNA from 1-mlsamples was purified and assayed for N3 and C4content (Table 1). During the first 85 min and at7 hr of Mg++ starvation, the fraction of RNApyrimidines derived from exogenous uracil was

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FIG. 3. Content ofradioactive RNA in exponentiallygrowing Aerobacter aerogenes, derived from an exog-enous precursor, as afunction ofincorporation time. Thesolid line gives the expected theoretical curve if thespecific activity of the synthesized RNA remains con-stant during the measurements and the mass doublingtime is 40 min. The observed radioactive RNA (countsper minute) as a function of time when the exogenousprecursor is labeled guanine, 40 ,ug/ml (0), or theexogenous precursor and sole source of carbon is uni-formly labeled glucose-C'4 (2 mg/ml) (@). The countsper minute for the three cases were normalized to becoincident at the latest time (100 min).

TABLE 1. Relative contributions ofexogenous uraciland glucose to pyrimidines of Aerobacter

aerogenes RNAa

Percentage of pyrimidine carbon derivedfrom uracil

Time ofincorporation During After Mg" starvation for

exponentialgrowth O hr 3 hr 7 hr 22 hr

35 31.6 34.8 14.7 32.8 18.350 32.0 36.2 22.0 33.4 26.670 36.5 16.4 34.8 22.385 30.0 34.8 21.3 33.0 21.6Avg 31.2 35.6 18.6 33.5 22.2

a The micromoles of uracil carbon (u) incor-porated into RNA of A. aerogenes 1033 are deter-mined from the amount of RNA-H3 and the spe-cific activity of the exogenous uracil-HP. Similarly,the micromoles of glucose carbon (g) incorporatedat the same time into RNA are determined. Thesum of these (G) gives the total carbon incorpo-rated. From the known base ratios of A. aerogenesRNA, it can be calculated that 19% of this carbon(G) is pyrimidine carbon. Therefore, u/0.19 G givesthe fraction of pyrimidine carbon derived fromexogenous uracil (see text).

similar to the value during exponential growth(31.2, 35.6, and 33.5% averages). However, at 3and 22 hr of starvation much more carbon forRNA pyrimidines was derived from endogenoussynthesis and much less from exogenous uracil(18.6 and 22.2% average). These values must beused to translate micromoles of uracil incor-porated into micrograms of RNA synthesized.The basis for ihese differences is not known, butpresumably they reflect specific changes in perme-ability or in the relative repression or inhibitionof biosynthetic enzymes involved in the synthesisof the pyrimidine nucleoside triphosphates.For the 1st hr or so of Mg++ starvation, the

incorporation of exogenous uracil into RNA pro-ceeded at rates comparable to the instantaneousrate of an exponentially growing culture (Ma-terials and Methods) and subsequently it con-tinued at an approximately linear rate for morethan 22 hr. The incorporations during a 90-mininterval commencing at 2.5, 7, and 22 hr areshown in Fig. 4. The slopes of the curves between40 and 80 min correspond to approximately one-third the instantaneous rate of the exponentiallygrowing populations after normalizing to a con-stant volume of culture (Materials and Methods);i.e., during this period, it requires approximately170 mn to synthesize an amount of RNA equalto that present at time zero.

Absolute rates of total RNA synthesis estimated

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N 1500

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FIG. 4. Rate ofRNA synthesis in Aerobacter aerogenes during Mg++ starvation. Bacteria were centrifuged afterthe indicated time ofstarvation and then resuspended in fresh MMG - Mg++ containing 5,6-uracil-H3 (18,ug/ml)and uniformly labeled glucose-C14 (2 mg/ml). Samples (0.2 ml) were acidified and acid-insoluble H3 was determinedfor the next 90 min. During this time, four samples (1.0 ml) were treated to purify RNA carbon free from all othercell carbon (see Appendix) in order to assess the relative contributions to RNA-pyrimidines of exogenous uracilcompared with glucose. These values (Table 1) were used to normalize each curve in order to translate uracil-H3incorporation into a relative rate ofRNA synthesis.

by tRNA accumulation. The rate of RNA synthe-sis can also be estimated by an entirely differentmethod. Since the ratio of synthesis of tRNA tothe synthesis of rRNA remains constant (Fig. 1),and since tRNA is stable (5), it is possible toestimate RNA synthesis by measuring the accu-mulation of tRNA during starvation. The amountof tRNA which accumulated during Mg++ star-vation was shown in Fig. 3 of the previous paper(5), and from this the relative content of tRNAper milliliter as a function of starvation time isplotted in Fig. 5. In agreement with the resultsdescribed above, the rate of synthesis soon be-came linear and remained constant for more than20 hr. From Fig. 5, it can be seen that this latterrate corresponds to a time of 110 min to synthe-size an Ro amount of RNA, i.e., between 2 and 22hr there is an almost 12-fold synthesis of theamount of tRNA present at the start of starva-tion.From these results, we concluded that for the

first 24 hr of Mg++ starvation the rates of synthe-

sis of both tRNA and rRNA are between one-third and one-half the instantaneous rate in thesame bacteria growing exponentially at the onsetof the starvation.

Rate ofDNA synthesis during magnesium star-vation. The net accumulation of DNA duringMg++ starvation of A. aerogenes was describedpreviously (5) and is shown in Fig. 6 (right). Theactual rate of synthesis is somewhat higher thanthat derivable from this curve due to a significantdegradation of part of the DNA during starva-tion. This breakdown reflects the presence, afterthe first few hours of Mg++ starvation, of a sub-population of dead cells (6) whose DNA is ap-parently degraded.

Unfortunately, exogenous thymidine incor-poration per unit of DNA synthesized decreasedprogressively with time of starvation. Thus, it isdifficult to estimate rates of DNA synthesis di-rectly by thymidine incorporation as was done forRNA synthesis by uracil incorporation (above),or for protein synthesis by amino acid incorpora-

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FIG. 5. Rate ofRNA synthesis in Aerobacter aerog-enes during Mg++ starvation. The relative content oftRNA (time 0 1.0) is estimatedfrom the counts perminute in the 4S region (tubes 26 to 34) of the sucrosegradient centrifugations shown in Fig. 3 of the previouspaper (5). Since the relative rates ofsynthesis of rRNAand tRNA are the same at all times (Fig. 1), the ratesderivedfrom this figure apply to both of these compon-ents.

tion (10). Instead, we estimated rates of DNAsynthesis by estimating the DNA which wouldhave been present had there been no degradation.The kinetics of DNA breakdown were very

similar at different times of Mg++ starvation.DNA remained acid-precipitable for close to 2.5hr after its synthesis (Fig. 6, left), and then ap-proximately 40% was degraded at an approxi-mately linear rate during the next 8 hr. [The initial2.5-hr lag probably reflects the minimal time be-tween the incorporation of thymidine into a repli-cating chromosome and the completion of thatreplication, subsequent segregation into an inertcell (6), and, finally, the appearance of acid-soluble products from breakdown of that chromo-some.] Subsequently, the decay was slower, withabout 7% of the original amount lost per 10 hr.As a control, when Mg-++ was present in themedium, DNA was completely stable for at leastfive generations of exponential growth. While thiswas a shorter time than was covered by the star-vation periods (Fig. 6, left), the labeled DNArepresented less than 4% of the total DNA afterfive generations of growth in unlabeled thymi-dine, whereas during Mg'- starvation DNAlabeled during the preceding exponential growthdecayed when it was still 30% or more of thetotal DNA.

Since the rate and extent of breakdown ofDNAmade at different times of Mg+-+ starvation ap-peared to be approximately the same, it was pos-sible to estimate the total DNA which wouldhave been present had there been no breakdownand, thereby, the rate of DNA synthesis. This

curve can be derived by estimating DNA con-tents after arbitrary times of Mg++ starvation:To do this, we made the following assumptions.(i) DNA synthesized at any time during thestarvation is broken down in a manner which isreflected by the curves given in Fig. 6 (left). Onthe average, the percentages of DNA made at agiven time, which is broken down after a certainnumber of hours, are: 7% between 10.5 and 20.5hr, 40% between 2.5 and 10.5 hr, and 0% be-tween 0 and 2.5 hr after its synthesis. (ii) Thedegradation between 2.5 and 10.5 hr, and duringthe subsequent period, is linear with time. Forexample, the fraction of DNA degraded 2.5 to10.5 hr after its synthesis is assumed to be (0 +0.40)/2 = 0.20, so that of this DNA, synthesizedover an 8 hr period, 80% remains. It appearsfrom Fig. 6 that the decay is not exactly linear inthis interval (2.5 to 10.5 hr), but the error intro-duced by this approximation is very small. (iii)The rate of DNA synthesis is constant. This isreasonable, at least after the first few hours, sinceboth the accumulation and decay rates are ap-proximately constant.For example, using these assumptions, if there

had been no decay, the amount of DNA at 20.5hr of starvation would be: D2o.5 = (9.3) (Do) =

(0.53) (Do) + (0.565) (10 hr) (D.) + (0.8) (8 hr)(D8) + (1.0) (2.5 hr) (D,), where Do = DNAper milliliter at time zero, of which (0.53) (Do)remains after 20.5 hr; D20.5 = net accumulatedDNA per milliliter after 20.5 hr = (9.3) (Do)from Fig. 6 (right); and D. = DNA synthesizedper milliliter per hour during Mg++ starvation.This gives D. = (0.60) (Do).

If there had been no decay, DNA per milliliterwould be (0.60) (20.5 hr) (Do) = (12.4) (Do)rather than the accumulated (9.3) (Do); i.e., 25%of the total DNA synthesized by the bacteria be-comes acid-soluble during the first 20.5 hr ofMg++ starvation.

This procedure gives as close an estimate ofthe rate of DNA synthesis as could be achievedwith the data available. After the first few hoursof Mg++ starvation, the rate of DNA synthesiswas quite constant (Fig. 6, right), such that anamount of DNA equal to that present at timezero was synthesized about every 130 min.

DISCUSSIONMeasurement of absolute rates ofRNA synthe-

sis. There are only three sources of carbon fornucleic acid or protein synthesis in a cell: (i) themajor carbon source(s) provided in the medium,e.g., glucose; (ii) specific precursor moleculesprovided in the medium, e.g., adenine for nucleicacids; (iii) products from breakdown of mole-cules in the cell. The last process "turnover" is

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FIG. 6. (left) Acid-precipitable counts per minute derived from methyl-thymidine-H3 or thymidine-2-C'4 inAerobacter aerogenes incubated for the times indicated on the abscissa in MMG (control) or in MMG - Mg++(bottom three frames) containing 200 ,ug/ml ofunlabeled thymidine. In the top twoframes, bacteria were exposed toeither thymidine-H3 or thymidine-C'4 forfive generations ofexponential growth and then washed and resuspended inthe above media. In the bottom two frames, bacteria were exposed to labeled thymidine for 20 min after 4 and 11hr ofMg++ starvation, respectively, and then centrifuged and resuspended in MMG - Mg++ containing 200 ,g/mlofunlabeled thymidine. Samples (0.1 ml) were brought to 5% trichloroacetic acid at 0 C and assayed for acid-pre-cipitable radioactivity. (right) Relative net concentration of DNA per milliliter of culture during Mg++ starvation(*), determined by colorimetric assay (5). CalculatedDNA per milliliter had there been no degradationi, as derivedfrom the net DNA per milliliter, and rates ofdegradation are shown by the dashed line.

the most difficult to measure, since exchange ofthe breakdown products may occur to little or noextent with precursors provided in the medium.The significance of turnover for these measure-ments is discussed below. [It is often not clearwhether the term "turnover" is used to refer tobreakdown of a macromolecule and simultaneousresynthesis of molecules within the same classfrom any source, or, as used by Mandelstam (9),"... reutilization of the breakdown products forsynthesis," i.e., a closed system. In this Discussionturnover will be used only in the latter, morerestrictive, sense.]

Carbon from products of ribosome decay. Sinceribosomes are decaying to acid-soluble productsduring Mg++ starvation, it is reasonable to sus-pect that a fraction of these products are reutilizedwithout exchanging with exogenous precursors;this would make any estimates of RNA synthesisbased only upon incorporation of exogenous

precursors appear too low. Fortunately, in arather unique way, the metabolic features ofMg++-deprived A. aerogenes provide a means toestimate the extent, if any, of this source of car-bon. tRNA continues to accumulate for morethan 22 hr of starvation (Fig. 5), and, further-more, this component appears to be stable (5).In contrast, after 21 hr of starvation, 75% of therRNA made during the preceding period of ex-ponential growth has been lost (5). However,when RNA is labeled only during exponentialgrowth, there is no change, within the limits ofthese measurements, in the amount of label intRNA during this time (the total radioactivitiesin tRNA differ by only 3% between 0 and 22 hrof starvation; see Fig. 6 in preceding paper).This is true in spite of the fact that the totaltRNA has increased about 12-fold during thistime. If one assumes that all RNA is derived froma common pool of nucleoside triphosphates, this

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evidence indicates that virtually none of the car-bon derived from ribosome breakdown finds itsway back into RNA. Thus, reutilization of rRNApyrimidine carbon, or its turnover, is negligiblein A. aerogenes under these conditions. The acid-soluble products must be excreted from the cells,and, indeed, products with characteristics ofnucleotides or their derivatives do accumulate inthe medium during Mg++ starvation (7). [It isinteresting that this capacity to reutilize break-down products of rRNA decay is species-specific.We have observed that, when Escherichia colistrain B is starved for Mg++, a significant frac-tion of the rRNA products are reutilized and theremainder are lost (unpublished data).]

Carbon from products of messenger RNA(mRNA) decay. A second source of "turnover"carbon is provided by the decay of mRNA. How-ever, in exponentially growing bacteria themRNA fraction represents only about 1% of thetotal RNA (2, 11). Thus, if the figure is compar-able in these cells, then even if the breakdownproducts were all reutilized (a very unlikely pos-sibility in view of the agreement between the hy-bridization and base ratio analyses in the abovepapers), the decrease in amount of exogenousprecursor incorporated by such displacementwould become insignfiicant after a short time.

Carbon from other acid-soluble compounds.Another complication results from the acid-soluble "pools" of base derivatives which slowlyfind their way into (or are in reversible equilib-rium with) the nucleoside triphosphates that areincorporated into nucleic acids. These intracellu-lar pools are not readily exchangeable with ex-ogenous labeled precursors; i.e., they are notchased out of the cells (see Fig. 7 in precedingpaper), and, thus, until maximally labeled, theydilute the specific activity of the nucleic acidslabeled by an exogenous precursor. Obviously,the relative size of these base pools and the speedwith which they equilibrate with the nucleosidetriphosphates will affect the specific activities ofthe latter and thus the legitimacy of translatingcounts per minute incorporated into milhimolesof nucleotide incorporated. Again, however, thetotal material involved in these pools is probablya small fraction of the total RNA (8), and thusany effects of dilution would be significant onlywhen measuring incorporation for short times.During Mg++ starvation, the acid-soluble nucleo-tide pools and their derivatives are even smallerthan in normal bacteria (7).The total effect of mRNA breakdown and

acid-soluble pools on guanine-H3 incorporationduring exponential growth is shown in Fig. 3.The solid curve represents the content of labeledRNA in the hypothetical case of an exponentially

growing population in which the specific activityof the newly synthesized RNA is constant as afunction of time. For guanine-IP incorporation,there appears to be an insignificant change withtime in the specific activity of the newly synthe-sized RNA. Uracil-IP incorporation is similar,whereas when the source of label (and all ex-ogenous carbon) is uniformly labeled glucose-C14, there is a measurable delay before the specificactivity of the RNA being synthesized reaches amaximum, i.e., before its carbon reaches thespecific activity of the exogenous carbon.Carbon from exogenous sources. Since, during

long periods of incorporation, contributions ofcarbon from turnover from pre-existing acid-soluble compounds to the synthesis of new RNAappears to be negligible in these cells, total syn-thetic rates can be estimated by the incorporationof carbon from exogenous sources. The relativecontributions of carbon from glucose and from aspecific precursor can change significantly duringa starvation, presumably by changes in permea-bility or in the extent of repression or inhibition,or both, of enzymes required for endogenous syn-thesis (Table 1). The extent of this effect will varyfor each precursor as a function of the degree towhich the processes are altered during this periodof metabolic imbalance.For this reason, RNA synthesis was measured

by incorporation of glucose carbon with or with-out simultaneous incorporation of nucleic acidbases into RNA. As indicated above, there is ameasurable delay after addition of uniformlylabeled glucose-C'4 to a culture before the specificactivity of the RNA being synthesized reaches amaximum. However, the total trichloroaceticacid-soluble carbon pool in E. coli is only 8% ofthe total cell carbon (13), and probably the bulkof this material represents end products of fer-mentation, compounds to be incorporated onlyinto other macromolecules (proteins, lipids, andpolysaccharides), plus small molecules with in-definitely long half-lives. The actual magnitude ofthis dilution in A. aerogenes can be estimated fromFig. 3 and is close to zero after 60 min of in-corporation.Another approach would be to determine the

specific activity of the nucleoside triphosphatepools of the cell in order to translate counts in-corporated into RNA into millimoles of nucleo-tide polymerized. However, besides the technicaldifficulties of characterizing such a small fractionof the bacteria, the final value represents a firtderivative of the amount of nucleic acid actuallyformed; e.g., if the specific activity of a givennucleoside triphosphate pool were changing con-tinuously with time, then one would need to makemany measurements during the time of study to

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determine the total amount ofnucleic acid synthe-sized.Recommended procedure for estimating rates of

RNA synthesis. As indicated above, the use of alabeled specific precursor, such as uracil, to esti-mate rates of RNA synthesis is complicated whenthe composition of the RNA is changing withtime. Thus, if possible, the following proceduresshould be used: (i) if it can be shown that oneRNA component remains stable during the periodin question, e.g., tRNA, then (ii) the accumula-tion with time of this component (Fig. 5) is ameasure of its rate of synthesis from all sources,and (iii) a specific precursor, such as uracil, canbe used to measure the relative rates of formationof rRNA and tRNA at various times (Fig. 1),and thus to derive the rate of rRNA synthesis.This protocol, which avoids the complicationsdiscussed above, could be used in the presentstudies because tRNA is stable during Mg++starvation.

Nucleic acid synthesis during Mg++ starvation.DNA synthesis continues at an approximatelylinear rate for at least 40 hr when A. aerogenes isincubated in a Mg++-deficient medium such thatan amount of DNA equal to that present at thestart of the starvation is produced every 2 hr.Using different methods, approximately the samerelative rates were found for the synthesis ofrRNA and tRNA, and in the next paper it will beshown that soluble proteins are also synthesizedat the same relative rates (10). The instantaneousrates of synthesis of all components in exponen-tially growing A. aerogenes is such that eachwould be doubled in 55 min (Materials and Meth-ods). Thus, with respect to these macromolecules,the culture continues to produce a normal com-position of protoplasm at a rate close to one-halfthat of the exponentially growing cells at the on-set of starvation. The significance of these ob-servations will be discussed elsewhere (6, 10).

ACKNOWLEDGMENTSThis investigation was supported by Public Health

Service research grant GM 09830-03 and traininggrant 5 TI Al 257-02 from the National Institutes ofHealth. Support for the Washington University Com-puting Facilities is provided by Grant G-22296 fromthe National Science Foundation.

LITERATURE CITED1. BELOZERSKY, A. N., AND A. S. SPIRIN. 1960.

Chemistry of the nucleic acids of microor-ganisms, p. 147-186. In E. Chargaff and J. N.Davidson [ed.], The nucleic acids, vol. 3.Academic Press, Inc., New York.

2. BOLTON, E. T., AND B. J. MCCARTHY. 1962. Ageneral method for the isolation of RNA com-plementary to DNA. Proc. Natl. Acad. Sci. U.S.48:1390-1397.

3. ENNIS, H. L., AND M. LUBIN. 1965. Pre-ribosomalparticles formed in potassium-depleted cells.Studies on degradation and stabilization. Bio-chim. Biophys. Acta 95:605-623.

4. GORINI, L., AND H. KAUFMAN. 1960. Selectingbacteria mutants by the penicillin method.Science 131:604-605.

5. KENNELL, D., AND A. KOTOULAS. 1967. Magne-sium starvation of Aerobacter aerogenes. I.Changes in nucleic acid composition. J. Bac-teriol. 93:334-344.

6. KENNELL, D., AND A. KOTOULAS. 1967. Magne-sium starvation of Aerobacter aerogenes. IV.Cytochemical changes. J. Bacteriol. 93:367-378.

7. KENNELL, D., AND B. MAGASANIK. 1962. The rela-tion of ribosome content to the rate of enzymesynthesis in Aerobacter aerogenes. Biochim.Biophys. Acta 55:139-151.

8. MCCARTHY, B. J., AND R. J. BRITTEN. 1962. Thesynthesis of ribosomes in E. coli. I. The incor-poration of C'4-uracil into the metabolic pooland RNA. Biophys. J. 2:35-47.

9. MANDELSTAM, J. 1960. The intracellular turnoverof protein and nucleic acids and its role in bi-ochemical differentiation. Bacteriol. Rev. 24:289-308.

10. MARCHESI, S. L., AND D. KENNELL. 1966. Magne-sium starvation of Aerobacter aerogenes. III.Protein metabolism. J. Bacteriol. 93:357-366.

11. MIDGELEY, J. E., AND B. J. MCCARTHY. 1962. Thesynthesis and kinetic behavior of deoxyribonu-cleic acid-like ribonucleic acid in bacteria. Bi-ochim. Biophys. Acta 61:696-717.

12. NOMURA, M., AND J. D. WATSON. 1959. Ribo-nucleoprotein particles within choloromycetin-inhibited Escherichia coli. J. Mol. Biol. 1:204-217.

13. ROBERTS, R. B., D. B. COWIE, E. T. BOLTON,P. H. ABELSON, AND R. J. BRITTEN. 1955. Studiesof biosynthesis in Escherichia coli. CarnegieInst. Wash. Publ. No. 607.

APPENDIX

Use of Filters to Separate Radioactive RNA fromother Cell Components

For the study of many metabolic problems, it isdesirable to use a nonspecific precursor from whichnucleic acids, proteins, and other cell components mayall become labeled. This necessitates the purificationof each class of macromolecule being studied freefrom all the others. The use of filters to separate pre-cipitable from soluble material has simplified thesefractionations enormously. For the extractions out-lined below, we have found glass-fiber filters (Micro-fiber Glass Prefilters, AP20, Millipore Filter Corp.,Bedford, Mass.; and Reeve Angel Glass Fiber Filters,Grade 934AH, H. Reeve Angel & Co., Inc., Clifton,N.J.) to be the most satisfactory.

Steudel and Peiser (11) first showed that RNA isquantitatively split to acid-soluble nucleotides bylong incubations in dilute alkali, whereas DNA re-mains acid-insoluble during this treatment. For thefractionation of cellular components, this differential

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lability to alkali was used by Schmidt and Thann-hauser (9) to separate RNA phosporus from DNAphosphorus. Unfortunately, under these conditionsthere is a progressive release with time of acid-solublefragments from proteins. Milder conditions of hy-drolysis can be used to minimize contamination of theRNA fraction by peptides. For example, Scott et al.(10) reported complete extraction of RNA from rattissues in 1 N NaOH at 22 C for 60 min, and laterinvestigators successfully extracted rat liver RNA, bytreatment for 1 hr at 37 C in 0.3 N NaOH (4), andtobacco leaf RNA, by extraction in 0.5 N KOH at 37C for 60 min (13). Alkaline hydrolysis of acid-pre-cipitated protein and RNA from A. aerogenes is shownin Fig. 1. The following procedures include a period ofalkaline hydrolysis which effectively separates RNAand protein into acid-soluble and acid-insoluble frac-tions, respectively.

Procedures. (RI) A small sample of material (0.2to 1.0 ml, depending on expected radioactivity, culturedensity, etc.) is mixed with an equal volume of 20%trichloroacetic acid in a test tube immersed in ice and10% trichloroacetic acid is added to bring the volumeto about 3 ml.

(R2) After 30 min or more, the resulting acid-pre-

cipitated material is collected on a filter presoaked (awash is sufficient) in 10% trichloroacetic acid at 0 C,and the filter is washed three times with 10% trichloro-acetic acid at 0 C and one time with 70% ethyl alcoholat 0 C.

(R3) The following lipid extractions are performedwith solvents maintained at 40 to 45 C: twice with 70%ethyl alcohol, twice with ethyl alcohol-diethyl ether(1:1), and once with diethyl ether. A chloroform ex-traction may be advisable for some tissues, especiallywhen extracting lipids which contain significantamounts of sphingomyelins (see reference 5).

(R4) The filter is then removed and placed upside-down in a 20-ml beaker to air-dry (about 10 min). A2-ml amount of 0.5 N NaOH at 37 C is added and thebeaker is kept at 37 C for exactly 40 min.

(R5) The alkaline hydrolysis is terminated by im-mersing the beaker in ice and adding 0.6 ml of 50%trichloroacetic acid, i.e., to a final concentration of 5%trichloroacetic acid.

(R6) After 30 min or more at 0 C, the contents ofthe beaker, including the filter, are poured onto a newfilter, and the filtrate, which contains acid-solubleRNA fragments, is collected into a large test tubewhich can be inside a vacuum flask under negative

5 10 15 20 25HOURS

FIG. 1. Hydrolysis ofacid-denaturedRNA andproteinfrom Aerobacter aerogenes in 0.5 NNaOH at 37 C. (top)Percentages ofradioactive protein, labeled with L-leucine-CQ4, and ofradioactive RNA, labeled with uracil-H', pre-cipitable in 5% trichloroacetic acid at 0 Cas a function ofincubation time. The insert shows valuesfor the first 2 hr.(bottom) Amount oforcinol-reactive and Folin-reactive material soluble in 5% trichloroacetic acid at 0 C; 100% =

the respective colors produced by completely hydrolyzed samples.

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pressure to accelerate the filtration. The beaker, theold filter, and the acid-soluble DNA and protein arewashed three or more times with 5% trichloroaceticacid at 0 C, and the filtrate is again collected into thesame tube. The total volume (about 15 ml) of thecombined filtrates is measured and recorded.

(R7) A suitable portion of the filtrate is preparedfor estimation of radioactivity (see below).

Acid filtrates can be counted conveniently in variousscintillation mixtures suitable for aqueous samplecounting. Bray's mixture (1) or a mixture containingthe thixotropic gel, Cab-O-Sil (7), were used success-fully.

If it is inconvenient to measure radioactivity in anacid ifitrate, it is possible to work exclusively withacid precipitates retained on filters. To do this, dupli-cate samples of cell material are treated in step R1;one sample is subjected to alkaline hydrolysis and thesecond is not. RNA radioactivity is then given by thedifference in radioactivities retained by the two filters.Because of significant quenching by trichloroaceticacid, it is important to treat all filters with a final washof ether or 0.1 N HCI, to remove traces of acid, beforeair-drying the filters for scintillation counting.

Extent of contamination and losses. After 40-minhydrolysis, no acid-insoluble RNA can be detected,whereas an insignificant fraction of the protein hasbeen lost (Fig. 1). However, since in many cells thereis three or more times as much protein as nucleic acid,the acid-soluble fraction was examined more critically.

Bacteria were grown on D-glucose-C'4 (uniformlylabeled) as sole source of carbon to label all cell car-bon. A preparative sample of these cells was treatedby the same procedures, except that KOH was usedin step R4 instead of NaOH, and, in step R5, 2.0 Nperchloric acid (PCA) at 0 C was used rather than 5%trichloroacetic acid. The resulting precipitate of pro-tein, DNA, and KC104 was centrifuged and then dis-carded. The supernatant liquid was again brought to0.5 N KOH, and, after centrifugation, the clear super-natant liquid of 0.5 N KOH, containing RNA mono-and oligonucleotides plus any contaminating carboncompounds, was incubated at 37 C for 18 hr to effectthe complete hydrolysis of oligonucleotides to the

t4

I0.e1

o

2'- and 3'-mononucleotides. The mixture was againacidified by addition of PCA and centrifuged to re-move KC104.The acid sample was then applied to a small char-

coal [Norit A-Celite (1:1)] column equilibrated with0.2 N PCA. Apparently, only heterocyclic and aro-matic compounds (such as nucleotides or aromaticamino acids) will'not elute from thecharcoalwith wateror acid (2, 3). When samples which initially had beenhydrolyzed (step R4) for 24 hr in 0.5 N KOH wereapplied to the column, a large fraction of the radio-activity eluted with water, thus indicating, as expected,considerable contamination by non-nucleotide ma-terial, presumably aliphatic amino acids or their pep-tides. Samples hydrolyzed (step R4) for 20 or 40 mincontained radioactivity of which 0.23 and 1.38%,respectively, eluted with water. Thus, after 40 min ofalkaline hydrolysis (step R4), less than 2% of theradioactivity in the acid-soluble fraction appears tobe nonaromatic; this, in itself, indicates insignificantcontamination from protein fragments.

However, it is still possible that the radioactivityretained by the charcoal contains compounds otherthan RNA nucleotides. To exclude this possibility,the adsorbed radioactivity was eluted with ethyl alco-hol-NH4OH (50:2) with a recovery of >95%. Theeluant was evaporated to dryness, and the residue wasresuspended in 0.12 ml of water. The four 2'- and 3'-ribonucleotides (Sigma Chemical Co., St. Louis, Mo.)were added as nonradioactive carriers, and two equalvolumes of this solution were spotted onto separateWhatman 3 MM papers for subsequent electrophore-sis in 0.05 M formate buffer, pH 3.5 (8). Both paperswere placed in the electrophoresis tank. However, onepaper was removed before application of voltagewhereas the second was subjected to 3 kv for 2.5 hr.Both papers were air-dried, the nucleotide spots wereidentified under an ultraviolet lamp, and strips ofpaper containing these spots were counted in a Van-guard Strip Counter (Autoscanner 880). All ob-servable radioactivity coincided with the position ofthe four nucleotides, and no radioactivity was ob-served to migrate when the electrodes were reversed(Fig. 2). Furthermore, the total radioactivities (from

h1500So1500 \

0

FIG. 2. Radioactivity in fraction R7 (Procedures) derived from bacteria grown on glucose-C'4 (uniformly la-beled) plus excess adenine, guanine, uracil, and cytosine. Peak at far right: before electrophoresis. Peaks to left:after electrophoresis. Electrophoresis in a partitioned tank with 0.05 M formate buffer (pH 3.5) in lower phases andVarsol in the upperphase to cool the paper (Whatman 3MM). Voltage = 3 kv, voltage gradient = 45 v/cm. Radio-activities estimated in a Vanguard Autoscanner 880 with: collimator, I cm; chart speed, 12 inches/hr; time con-

stant, 30 sec. Note the more prominent GMP peak which reflects its more elevated endogenous synthesis comparedwith the synthesis ofthe other nucleotides when bacteria are grown in the presence ofnucleic acid bases.

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the area under the curves) were estimated and the sumof that of the four nucleotides agreed with that of thecontrol spot within the accuracy (±15%) attainablewith the use of this type of strip scanner [see formulafor standard error in a recent paper by Johnson (6)].Thus, it is possible that a small fraction of the radio-activity is non-nucleotide material which, however,must also be adsorbed to charcoal and remain at theorigin under these conditions of electrophoresis.

Regarding losses, Venkateraman and Lowe (12)reported significant losses of rat liver RNA duringlipid extractions with ethyl alcohol of the trichloro-acetic acid-precipitated material. We have not ob-served these losses of RNA from A. aerogenes. How-ever, often as much as 5% of the RNA radioactivityis not recovered after the alkaline hydrolysis of ma-terial retained on the filter. Presumably this reflectssome variability with respect to penetration time forsaturation of the filter, nonspecific adsorption of largeroligonucleotides, or other unknown factors.

With the reservations just considered, the proce-dures, which include about 14 extraction steps, providea rapid method for estimating radioactivity in micro-gram amounts of RNA.

LIrERATURE CITED1. BRAY, G. A. 1960. A simple efficient liquid scintil-

lator for counting aqueous solutions in a liquidscintillation counter. Anal. Biochem. 1:279-285.

2. CRANE, R. K., AND F. LIPMANN. 1953. The effectof arsenate on aerobic phosphorylation. J.Biol. Chem. 201:235-243.

3. FISKE, C. H. 1934. Nature of depressor substanceof blood. Proc. Natl. Acad. Sci. U.S. 20:25-27.

4. FLECK, A., AND H. N. MuNRo. 1962. The pre-cision of ultraviolet absorption measurementsin the Schmidt-Thannhauser procedure fornucleic acid estimation. Biochim. Biophys. Acta55:571-583.

5. HuTCHISON, W. C., AND H. N. MuNRo. 1961. Thedetermination of nucleic acids in biologicalmaterials. A review. Analyst 86:768-813.

6. JOHNSON, M. J. 1965. Optimum operation of re-cording count rate meters for radioactive paperchromatograms. J. Chromatog. 20:100-106.

7. KREISBERG, R. A., AND J. R. WILLIAMSON. 1964.Metabolic effects of glucagon in the prefusedrat heart. Am. J. Physiol. 207:721-727.

8. MARKHAM, R., AND J. D. SMITH. 1952. The struc-ture of ribonucleic acid. I. Cyclic nucleotidesproduced by ribonuclease and by alkaline hy-drolysis. Biochem. J. 52:552-557.

9. SCHMIDT, G., AND S. J. THANNHAUSER. 1945.Method for determination of desoxyribonucleicacid, ribonucleic acid, and phosphoproteins inanimal tissues. J. Biol. Chem. 161:83-89.

10. SCOTT, J. F., A. P. FRACCASTORO, AND E. B. TATr.1956. Studies in histochemistry. I. Determina-tion of nucleic acids in microgram amounts oftissue. J. Histochem. Cytochem. 4:1-10.

11. STEUDEL, H., AND E. PEISER. 1922. Uber die Hefe-nucleinsaure. III. M. Heilung. Z. Physiol.Chem. 120:292-295.

12. VENKATARAMAN, P. R., AND C. U. LOwE. 1959.Effect of ethanol on rat liver ribonucleoproteinpreviously exposed to cold trichloracetic acid.Biochem. J. 72:430-435.

13. WOLLGIEHN, R., AND B. PARTHIER. 1964. Quanti-tative determination of ribonucleic acid andprotein in leaves. Flora 154:325-348.

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Magnesium Starvation of Aerobacter aerogenes · Magnesium Starvation of Aerobacter aerogenes II. Rates of Nucleic Acid Synthesis andMethodsfor Their Measurement DAVID KENNELLAND ANGELIKI