JOURNAL OF BACTERIOLOGY, Feb. 1980, p. 4564630021-9193/80/02-0456/08$02.00/0

Vol. 141, No. 2

Structural Specificity of the Spermidine Requirement of anEscherichia coli Auxotroph

CAROLINE M. JORSTAD, JOHN J. HARADA, AND DAVID R. MORRIS*Department ofBiochemistry, University of Washington, Seattk, Washington 98195

A homologous series of spermidine analogs was synthesized with the generalstructure NH3+(CH2),NH2+(CH2)3NH3+, where spermidine has n = 4. The influ-ence ofthese compounds on growth and on the syntheses of protein and messengerribonucleic acid was examined in a spermidine auxotroph of Escherichia coli. Allof the homologs tested were taken up by the cells to an intracellular levelequivalent to the level of spermidine which gives optimal growth. With increasingchain length of the homologs, there was reduced ability to stimulate growth. Thehomologs with n = 7 and n = 8 were essentially inactive. A similar specificity wasobserved when the ability of the homologs to restore the rates of protein andmessenger ribonucleic acid chain elongation was compared to that of spermidine.These results suggest that a definite spatial arrangement of the amino groups ofspermidine is required for productive interaction at its intracellular site(s) ofaction.

Wild-type Escherichia coli growing in mini-mal medium contains milimolar intracellularconcentrations of spermidine [NH3+(CH2)4-NH2+(CH2)3NH3+] and its precursor putrescine[NH3+(CH2)4NH3+] (30). Mutants of E. coli thatare defective in most of the enzymatic steps ofputrescine and spermidine biosynthesis havebeen isolated (2, 7, 17, 27). The physiology ofthese polyamine-deficient cells has been of in-terest in recent years (16). Starvation for polya-mines reduced the growth rate of the mutantcells and produced an identical reduction in therates of RNA and protein syntheses (14). Inhi-bition ofRNA and protein syntheses was due todecreased polymerization rates rather than tospecific effects only on initiation (9, 15). Therate ofDNA replication fork movement was alsoslowed during polyamine deficiency (6). As anapproach to further define the biological role(s)of spermidine, we examined the structural spec-ificity of this requirement for optimal growthand macromolecular synthesis. In the work re-ported here, we synthesized a series of spermi-dine homologs of the general structureNH3+(CH2),NH2+(CH2)3NH3+. All of the homo-logs tested were taken up by the mutant cells,and specificity was observed for support ofgrowth and the rates of synthesis of RNA andprotein.

MATERIALS AND METHODSSpermidine homologs. The homologs of spermi-

dine used in this study (Table 1) were synthesized bythe procedure of Israel et al. (8). The appropriatediamine was reacted with redistilled acrylonitrile(Matheson, Coleman & Bell). The monocyanoethyl

derivatives synthesized by this procedure were col-lected by distillation, and their purities were tested bythin-layer chromatography on cellulose plates (13).Reduction was carried out in a Parr hydrogenator byusing Raney sponge nickel 28 (W. R. Grayson Co.) asa catalyst. After distillation, the product was dissolvedin absolute ethanol, and the hydrochloride salt wascollected after bubbling HCI gas through the solution.The identities of the products were confirmed byelemental analysis (Table 1). No ninhydrin-positivecontaminants were detected by thin-layer chromatog-raphy (13) or by ion-exchange chromatography (19;see below). The diamine starting materials were pur-chased from the following sources: 1,5-dianinopentaneand 1,8-diaminooctane, Matheson, Coleman & Bell;1,6-diaminohexane, Ames Laboratories; and 1,7-dia-minoheptane, Aldrich Chemical Co. The 1,5-diamino-pentane and 1,6-diaminohexane were distilled beforeuse; the others were used directly.

Bacterial strain and culture conditions. E. colistrain DK6 was constructed by transduction of a speAmutation (biosynthetic arginine decarboxylase) intoE. coli strain AS19 (18). Strain AS19 was originallyisolated for its sensitivity to actinomycin D, but wassubsequently found to be sensitive to a spectnrm ofantibiotics, including fusidic acid, rifampin, and strep-tolydigen (23). Cultures were starved by ovemightgrowth in the absence of polyamines, as previouslydescribed (18). The biochemical basis of this starvationhas been discussed extensively (16, 17). The starvedcultures were diluted and were grown with shaking at37°C for at least three generations with or withoutpolyamine supplementation in the culture mediumdescribed by Neidhardt et al. (21) supplemented with0.2% glucose and a synthetic amino acid mixture (22).Routine polyamine supplementation was as follows:spermidine, 0.4 to 0.6 yig/ml; AP5, 0.3 to 0.6 jig/ml;AP6, AP7, and AP8, 25 to 100 jig/ml. These polyamineconcentrations in the culture medium resulted in com-parable intracellular levels (see below).

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TABLE 1. Spermidine homologs testedElemental analysis (%)

Designation Structurea C N H Cl

Calcu- Ob- Calcu- Ob- Calcu- Ob- Calcu- Ob-lated served lated served lated served lated served

AP5 NH2(CH2)5NH(CH2)3NH2 35.96 35.66 15.73 15.55 8.99 8.68AP6 NH2(CH2)6NH(CH2)3NH2 38.43 38.44 14.95 14.78 9.25 9.42 37.37 37.31AP7 NH2(CH2)7NH(CH2)3NH2 40.68 40.54 14.24 14.09 9.49 9.28 35.59 35.49AP8 NH2(CH2)8NH(CH2)3NH2 42.72 42.61 13.59 13.50 9.71 9.83 33.98 33.91

aThe amines were prepared as the hydrochloride salts.

Cellular composition. The cellular contents ofprotein, RNA, and DNA were analyzed as previouslydescribed (18). For measurement of polyamines, 30-mlcultures at a density of 10" cells per ml were pouredonto ice with 10 mM (final concentration) NaN3 tostop growth and to prevent acetylation of spermidine.The cells were collected by centrifugation. Polyaminepools were extracted as previously described (19). Ourprocedure for analysis has been described previously(19) and was modified by using a new resin system(29). All of the triamines were separated in this ana-

lytical system with appropriate alterations in the ionicstrengths of the elution buffers (Table 2).Polysome analysis. Polysomes were extracted and

analyzed as previously described (9) with the followingmodifications. The culture medium was changed tothat described above (21, 22). The efficiency and re-

producibility of lysis were improved by the addition ofsodium deoxycholate to 0.05% (wt/vol) after thefreeze-thaw regimen.

/1-Galactosidase induction. Growth of the cells,enzyme induction with isopropyl thiogalactoside, sam-pling into streptolydigen-containing medium, and the,B-galactosidase assays were perfonned exactly as pre-viously described (15). Enzyme units were defined as

nanomoles of o-nitrophenyl galactoside hydrolyzedper minute and were normalized to 1 ml of culture atan absorbance at 450 nm of 1.0.

RESULTS

Polyamine content, growth, and macro-molecular composition. Strain DK6 was

grown in the presence of each of the spermidinehomologs (Table 1). The levels of cell-associatedputrescine, spermidine, and the particular hom-olog in question were measured (Table 3). Pu-trescine was not detected under any of thegrowth conditions, which is consistent with themutational defect in arginize decarboxylase (18).Cells grown in the absence of a polyamine ad-dition contained approximately 10% of the nor-

mal level of spermidine, as found previously (18).All of the spermidine homologs were taken upby the cells, although there were considerabledifferences in the efficiency of uptake. AP5 andspermidine reached equivalent intracellular lev-els at similar exogenous concentrations. How-ever, the cells concentrated AP6, AP7, and AP8much less efficiently; exogenous levels of at least

TABLE 2. Buffers and elution times for amineanalysisElution time (min)

Secondbuffere Putres- Spermi- APS AP6 AP7 AP8

cine dine

1.5 N K+ 24 49 552.4N K+ 22 44 54 634.0N K+ 22 44 62

'All buffers were pH 5.6 and contained a molar ratio ofKCI to potassium citrate of 26.7. Buffers are designated bytheir total normality of potassium ion. In all analysis runs, thefirst buffer contained 0.5 N KW. At 30 min, a change was madeto the indicated second buffer. The elution times are from thestart of the first buffer.

TABLE 3. Polyamine content and growth ratePolyamine content

(nmol/mg ofconcn (,Ag/ prti)"b rotPolyamine ml of cul- protein rathaddeda ture me- r

dium) Spermi- Homologdine

None 5.2 0.95Spermidine 0.4 47 2.00AP5 0.6 3.1 52 1.62AP6 100 2.3 57 1.36AP7 50 2.1 44 1.04AP8 25 1.7 37 0.91

"See text for details of growth conditions.The putrescine limit in all cultures was <0.2 nmol/mg.

c Expressed mass doublings per hour.

25 ltg/ml were required to give intracellular pol-yamine concentrations similar to those obtainedwith spermidine at a concentration of 0.4 ,ug/ml.The polyamine determinations reported in Ta-ble 3 were performed on acid-hydrolyzed sam-ples. Omitting the acid hydrolysis did not alterthe results for spermidine, AP5, and AP8. Thevalues for AP6 and AP7 in unhydrolyzed sam-ples were lower by 40 and 15%, respectively,suggesting partial derivatization of these com-pounds by the cells.Although the above results suggested that

similar levels of cell-associated spermidine andits homologs could be reached by providing ap-propriate concentrations in the culture medium,the cellular location of the various triaminesmight differ. Spermidine uptake by E. coli con-

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sists of two components, a rapid process notdependent on metabolic energy, which is com-plete in less than 2 min, and a slower, energy-dependent uptake (25). The first process was

thought to be simple adsorption to the cell sur-

face since it occurred at 00C and the adsorbedmaterial was rapidly exchangeable (25). To testthe extent of adsorption in these experiments,starved cells of strain DK6 were treated with 10mM NaN3 at either 37 or 00C and then exposedto spermidine or its homologs for 5 min at theconcentrations indicated in Table 3. In all cases,the levels of cell-associated triamines were lessthan 10% of those reported in Table 3. Thus, thelevels attained in growing cells were not duesimply to adsorption to the cell surface. In ad-dition, an assessment of the distribution of sper-midine and its homolog AP6 in cell extracts wasmade by differential centrifugation. Strain DK6was grown in the presence of either 0.3 iLg ofspermidine per ml or 25 jig of AP6 per ml. Thetwo cultures were treated with NaN3 and mixed,and the cells were harvested. Lysates were pre-pared by the lysozyme-freeze-thaw method (seeabove for polysome analysis) without the use ofdetergents, treated with DNase, and fraction-ated into a low-speed (30,000 x g, 30 min) pelletand a high-speed (70,000 x g, 17 h) upper super-natant, lower supematant, and pellet. The dis-tribution ofcellular spermidine in these fractionswas 7, 22, 38, and 33%, respectively. The distri-bution of AP6 was very similar (7, 22, 44, and27%, respectively). Although these results do notnecessarily reflect intracellular localization (seebelow), it seems clear that the spermidine hom-olog AP6 was internalized by the cells and was

distributed similarly to spermidine in the frac-tions separated by differential centrifugation.The growth rates of strain DK6 in the pres-

ence of similar cellular levels of the varioustiamines are compared in Table 3. In the ab-sence of added amines, the growth rate was

twofold lower than that observed in the presenceof spermidine, as reported previously (18). Cellsgrown with putrescine in the culture mediumgrew at a rate identical to that observed withspermidine (specific growth rate, 2.0 doublingsper h) (data not shown). The longer-chainedtriamines (AP7 and AP8) did not signifcantly

enhance growth, although the intracellular con-centrations were similar to the spermidine levelsgiving optimal growth. AP7 and AP8 were testedin the range from 10 to 200 ,ug/ml and had no

effect on the growth of the starved cells, exceptfor a significant inhibition at the higher concen-

trations of AP8. AP5 and AP6, also at intracel-lular levels equivalent to spermidine levels inspermidine-supplemented cultures, gave inter-

mediate enhancement ofgrowth, with AP5 beingmore effective than AP6 (Table 3). Raising theconcentration of AP5 in the medium (10 ,ug/ml)increased the growth rate of the cells signifi-cantly (specific growth rate, 1.8 doublings perh). Thus, AP5 at sufficiently high intracellularconcentrations seems to be nearly as effective asspermidine in fulfilling the polyamine require-ment of this auxotroph. AP6 was not tested atconcentrations higher than the 100 yig/ml re-ported in Table 3.

It is clear that none of the homologs can fullyreplace the spermidine requirement at equiva-lent intracellular concentrations. However,quantitative interpretation of these differencesin growth rates is difficult since supplementationwith the homologs reduced the basal cellularspermidine content as much as threefold belowthe level in starved cells (Table 3).

In these experiments, exponential growth wasroutinely observed over an 80- to 160-fold in-crease in cell mass after addition of the homolog.In other experiments, where subculturing wascontinued over a period of several days, nochange in growth rate was observed until revert-ants prototrophic for putrescine synthesis ap-peared in the cultures. Hence, it appears thatthe cells maintain steady-state growth indefi-nitely in the presence of the homologs, albeit atlower rates.The macromolecular composition of strain

DK6 grown in the presence of the various tria-mines is shown in Table 4. For the measure-ments described here, cells were starved for pol-yamines as described above. The cultures werethen grown with the indicated polyamine sup-plementation for at least an 80-fold increase inmass before sampling. Duplicate measurementsmade approximately one mass doubling apartagreed within experimental error, indicating thata steady-state situation existed during the courseof the experiment. The RNA content of strainDK6 was unaltered by polyamine starvation orby growth in the presence of the homologs. Theinvariance in the RNA/protein ratio during pol-

TABLE 4. Macromolecular composition

Polyamine RNA cofncn DNA concn Protein concnaddeda (Ug/pg Ofpt (g/109 cells) (lg/109 cells)tein)

None 0.47 19 400Putrescine 0.49 21 350Spermidine 0.48 25 630AP5 0.50 19 480AP6 0.48 15 315AP7 0.50 18 430AP8 0.50 18 490

a See text for details of growth conditions.

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SPERMIDINE SPECIFICITY 459

yamine limitation of growth has been describedpreviously (18). This is a reflection of the factthat cellular ribosome content is constant duringpolyamine starvation, but the rate of proteinsynthesis per active ribosome decreases (9). Inaddition, the DNA contents of the cells showedonly minor differences under the various growthconditions. However, examination of cell size(i.e., protein content) revealed an interestingproperty. Although there were only small vari-ations in the protein contents of starved cells,putrescine-grown cells, and cells grown in thepresence of the homologs, the spermidine-growncells were larger by about 50%. Strain DK5,which is the parent of DK6 and is prototrophicfor polyamine synthesis (18), had a protein con-tent of about 400,ug/109 cells under these growthconditions. Thus, stimulation of growth by sper-midine in the absence of intracellular putrescineled to an increase in cell size.Ribosome distribution. Control cultures

grown in the presence of putrescine or spermi-dine were labeled with [14C]uracil. Starved cul-tures or cultures grown with the spermidinehomologs were labeled with [3H]uracil and thenmixed with 14C-labeled control cells. Lysateswere analyzed by sucrose gradient centrifuga-tion. To illustrate the type of profiles observed,an experiment with AP8 is shown in Fig. 1. Thepolysome patterns from control and AP8-growncells were similar (Fig. 1A). All ofthe radioactivematerial in the polysome region of the gradientwas converted to 70S monosomes by treatmentwith pancreatic RNase (data not shown). Theregion consisting of subunits and monosomes isdisplayed in more detail in Fig. 1B. Other thana small increase in the ratio of subunits to mon-osomes in the AP8-grown cells, there were nomeaningful differences in this region of the gra-dient between the two cultures. The data fromexperiments in which each of the homologs wastested are summarized in Table 5. As the lengthof the homolog increased, there was a slightdecrease in the amount of ribosomal material inpolysomes and a corresponding increase in thesubunit region. Using these data on the fractionof ribosomes in polysomes, together with thecellular growth rates (Table 3), the cellularRNA/protein ratio (Table 4), and the value of87% of total RNA as rRNA (18), we calculatedpolypeptide chain elongation rates for cells un-der the various culture conditions (Table 5). Aswas found previously (9, 15), starvation for pol-yamines reduced the rate of polypeptide chainelongation in a manner similar to the decreasein growth rate. The spermidine homologsshowed a specificity similar to that observed forrestoration of growth (Table 3). Thus, AP5 and

0

F RACTION

FRACTION

FIG. 1. Sucrose density gradients comparing pat-terns ofpolysomes and ribosomal subunits from cellsgrown in the presence of spermidine and AP8. Thecells were grown as described in the text. l4C]uracil-labeled cells (0) grown in thepresence ofspermidinewere mixed with [3H]uracil-labeled cells (0) grownwith AP8 supplementation. (A) For analysis ofpoly-somes, centrifugation was for 145 min at 36,000 rpmin a Beckman SW41 rotor, using a 10-ml gradient of15 to 30% sucrose over a 1-ml cushion of45% sucrose;6.15 x 106 cpm of 14C and 1.18 x Itt cpm of 3H wereapplied to the gradient. (B) For analysis of mono-somes and subunits, centrifugation was for 185 minat 45,00X rpm in a Beckman SW50.1 rotor, using a4.6-ml gradient of 15 to 35% sucrose over a 0.2-mlcushion of80% sucrose; 7.16 x 104 cpm of 14C and 5.64x 105 cpm of3H were applied to the gradient.

AP6 stimulated the elongation rate, but wereless effective than spermidine. AP7 and AP8were essentially inactive.Induction of enzyme-forming capacity.

We previously showed that polyamine starva-tion slowed the rate of fl-galactosidase messen-ger chain elongation (15). To estimate the influ-ence of the spermidine homologs on this param-eter, we measured the time from the addition of

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TABLE 5. Ribosome distributions and calculatedpolypeptide elongation rates

Ribosome distributionbPolyamine Elongationaddeda Poly- Mono- Subunits ratec

somes somes

None 69 12 19 8.2Spermidine 71 12 17 14.9AP5 66 12 22 13.7AP6 66 14 20 12.0AP7 61 12 27 8.8AP8 59 11 30 8.4

aSee text for details of growth conditions.b Expresed as percentage of total ribosomal material.'Amino acids per second per growing chain. Calculated as

described by Forchhammer and Lindahl (4) by using theformula [(P/120) x p x Ln2]/[(R/1.6 x 10w) x f], where P andR are protein and rRNA contents of the cells in microgramsper milligram (dry weight), respectively, i is the specificgrowth rate in mass doublings per hour, and f is the fractionof ribosomes active in protein synthesis. The value for f wascalculated by amuming that all of the polysomes and one-halfof the monosomes were active in protein synthesis.

inducer to the first appearance of active messagefor ,B-galactosidase. After induction, cultureswere sampled at timed intervals into solutionscontaining the antibiotic streptolydigen to blockfurther RNA synthesis (1, 15, 23). The sampleswere further incubated to allow expression ofcompleted message, and /i-galactosidase wasthen assayed (Fig. 2). The induction lags instarved and unstarved cells were similar to thoseobserved previously (15). The four homologswere less effective than spermidine in shorteningthe lag period; their effectiveness decreased withincreasing chain length.To interpret these results, it is necessary to

know whether polyamine starvation or supple-mentation of the cells with the homologschanged the rate of action of the inducer. Inwild-type E. coli, induction ofthe lactose operonbegan simultaneously with the addition of iso-propyl thiogalactoside (10) and was not influ-enced by polyamine starvation (15). This wastested in cells grown in the presence ofAP8 (Fig.3). In this experiment, transcriptional initiationwas blocked with rifampin at various times be-fore and after addition of isopropyl thiogalacto-side at zero time. The cultures were then incu-bated for completion and expression of previ-ously initiated mRNA and then assayed for ,B-galactosidase. In the AP8-grown culture, as wasfound previously for polyamine-starved cells(15), the accumulation of rifampin-resistant, en-zyme-forming capacity extrapolated back to zerotime. Identical results were obtained with AP5,AP6, and AP7. Thus, growth in the presence ofthe homologs introduced no lag in inducer ac-tion.

Since the inducer acts essentially instanta-

8

L4J6V0

0qKf

2

40 80 120 160

TIME OF INDUCTION (sec)

FIG. 2. Kinetics ofproduction ofstreptolydigen-re-sistant, enzyme-forming capacity. Cultures were

grown and induced with isopropyl thiogalactoside atzero time (see text). At the indicated times, samples

were taken into streptolydigen, incubated for 20 minat 35°C for expression of enzyme-forming capacity,and assayed (15). The basal, uninduced levels (0.88to 1.46) were subtracted to allow better comparison ofthe curves. Curve 0 is for the starved culture, curve 4is for the spermidine-supplemented culture, andcurves 5 through 8 are for cultures grown with hom-ologs AP5 through AP8.

neously under the conditions tested in this re-search, it is possible to estimate the rates ofmessage elongation from the induction lags (15).The average values for the induction lags fromthree independent experiments are listed in Ta-ble 6. A length for the lacZ gene of 3,063 nucleo-tides was estimated from the length of the poly-peptide chain (5), and rates ofmRNA elongationwere calculated. The values of 28 and 42 nucleo-tides per s in starved and unstarved cells, re-

spectively, were in agreement with previouslyreported values (15). As was observed qualita-tively in Fig. 2, the values for the spermidinehomologs were intermediate between the twoextremes.

DISCUSSIONStrain DK6 requires spermidine or its biosyn-

thetic precursor putrescine for optimal growth.The object of the present study was to examinethe'structural constraints of this requirement bymodifying the spermidine molecule. In order forthese results to be meaningful, the physiologicaleffects of the spermidine homologs would haveto be compared with the effects of spermidine at

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SPERMIDINE SPECIFICITY 461

1.5-

()1.0

0.5.

-60 -40 -20 0 20 40 60TIME OF INDUCTION (sec)

FIG. 3. Formation ofrifampin-insensitive enzyme-

forming capacity in cells grown in the presence ofAP8. Rifampin (60 ltg/ml) was added to samples ofthe culture at the times indicated on the abscissa,and isopropyl thiogalactoside was added at zero

time. The samples were then incubated at 35°C for 15min for expression of enzyme-forming capacity andassayed for f3-galactosidase.

TABLE 6. /8-Galactosidase induction lags andcalculated mRNA elongation rates

Polyamine Lag Elongation rateaddeda (8)b (nucleotides/s)'

None 110 28Spermidine 73 42AP5 85 36AP6 95 32AP7 98 31AP8 104 29

a See text for details of growth conditions.b The values were derived from back extrapolation

of the fmal slopes of induction curves such as thoseshown in Fig. 2. Values are the averages of threeindependent experiments.

'Calculated as described in the text.

equivalent intracellular concentrations. Thiswas achieved by appropriately adjusting theconcentrations of the polyamines added to theculture medium. Our results revealed a definitespecificity for growth. Lengthening the C4 meth-ylene chain of spermidine decreased the growth-promoting activity of the triamine, with the C7and C8 compounds being essentially devoid ofbiological activity. When the influence of thetriamines on protein and RNA syntheses was

examined, a qualitatively similar activity serieswas observed. This specificity could have arisenin one oftwo ways. First, there may be a require-ment for a certain spatial arrangement of theamino groups of spermidine for productive in-teraction at its cellular site of action. Alterna-tively, the least active homologs might be una-vailable for interaction at the appropriate cellu-lar sites, either because of covalent conjugationor because of an altered intracellular localiza-tion. Neither of the latter two possibilities seemslikely. Analysis of hydrolyzed and unhydrolzyedsamples revealed no correlation between appar-ent degree of conjugation and ability to restoregrowth. Examination of the distribution of sper-midine and AP6 by differential centrifugation ofcell lysates failed to detect a significant differ-ence. It should be emphasized that redistribu-tion of the triamines would be expected to anundefined extent in the combined lysate (25).However, the important point is that the twocompounds redistributed identically; AP6showed no evidence of sequestration by or selec-tive affinity for subcellular components. In ad-dition, the homologs showed no specificity foradsorption to the cell surface. Thus, the mostlikely explanation of our results at present isthat the specificity for the triamines arose at thesite(s) of spermidine action.

It is of interest that a qualitatively similaractivity series was observed for the influence ofthe triamines on growth and on the syntheses ofRNA and protein. These results could be con-sistent with the triamines exerting their effecton these three parameters at a single intracel-lular site of action. As discussed previously (15),this site could be either protein or RNA synthe-sis, because of the coupling between the twoprocesses. Thus, stimulation of either transcrip-tion or translation could affect the other proc-esses. Alternatively, spermidine may exert itsgrowth-promoting activity through interactionat multiple sites in the cell. In this case, thesimilar specificity for stimulation of protein andRNA syntheses might suggest a unifying fun-damental interaction of spermidine in the twoprocesses, for example binding of the triamine tonucleic acids (see below).The natural occurrence of sym-homospermi-

dine (11) and of sym-norspermidine (AP3) (3, 24,31) leads one to suspect that there is a certainlatitude in the spatial arrangements ofthe aminogroups of spermidine as far as its biological func-tion is concerned. The fact that AP3 was equiv-alent to spermidine in restoring the growth ofpolyamine-starved E. coli cells was consistentwith this possibility (18). This paper describesthe first systematic study of this question. It

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seems clear that extending the C4 methylenechain to C5 and longer reduces the biologicalactivity of the molecule. This may be related tothe mode of binding of the spermidine moleculeto DNA. Liquori and co-workers (12) proposeda model for spermidine binding to DNA in whichN5 and N'0 interact with phosphate residues onopposite sides of the narrow groove and N' andN5 bind to phosphates on the same strand.Lengthening the 04 methylene chain would alterthe interactions across the groove and thusmight give rise to the altered biological proper-ties of the homologs.Although this paper has addressed specifically

the function of spermidine, the intracellular con-centration of putrescine, its biosynthetic precur-sor, can be higher than the concentration of thetriamine by 3- to 10-fold in wild-type E. coli(30). Despite this high level of putrescine, thereare several lines ofevidence suggesting that sper-midine is the physiologically more important ofthe two in terms of cell growth. Strain DK6grows at the wild-type rate with undetectableintracellular levels of putrescine (Table 3). Thissame conclusion was reached previously withanother mutant strain of E. coli (17). Althoughthe growth ofE. coli in the absence of putrescineseems normal, there appear to be some physio-logical abnormalities. The protein content ofwild-type E. coli and of mutant cells grown inthe presence of putrescine is considerably lessthan that when growth of the mutant is stimu-lated by spermidine. This suggests a possiblerole for putrescine or for the ratio of putrescineto spermidine in determining cell size at divisionwhen the cultures are growing at wild-type rates.On the other hand, at suboptimal growth rates(either starved cells or growth with the homo-logs) cell size is normal in the absence of putres-cine. Munro and Bell (20) showed that withauxotrophs in culture media of low osmolarity,putrescine allowed wild-type growth, whereascultures containing spermidine grew moreslowly. It is not known whether this possible roleof putrescine in osmotic balance is related to theeffect on cell size. In any event, these results arehighly suggestive of a physiological role for pu-trescine different from that of spermidine.Can putrescine alone fulfill the polyamine re-

quirement in E. coli' The answer is probablyno, at least not with equal efficiency. Mutantswith a complete block in S-adenosylmethioninedecarboxylase have no detectable intracellularspermidine and a 70% increase in putrescine overthe wild-type level (27). These celLs show a re-duction in growth rate of approximately 25%,which is restored to nornal by the addition ofspermidine. Thus, it appears at present that

putrescine and spermidine may play their ownspecific roles in E. coli, with some overlappingspecificity. However, this situation may differconsiderably with different microbial species,since there is a wide range of phylogenetic vari-ation in amine contents (28).

ACKNOWLEDGMENTSWe thank P. A. Whitney for thoughtful comments on the

manuscript. J.J.H. derived support from Public Health Servicetraining grant GM 07270 from the National Institutes ofHealth. This work was also supported in part by Public HealthService research grant GM 13957 from the National Institutesof Health.

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3. De Rosa, M., S. De Rosa, and A. Gambacorta 1976.Occurrence and characterization of new polyamines inthe extreme thermophile Caldariella acidophila. Bio-chem. Biophys. Res. Commun. 69:253-261.

4. Forchhammer, J., and L Lindahl. 1971. Growth rateof polypeptide chains as a function of the cell growthrate in a mutant of Escherichia coli 15. J. Mol. Biol.55:563-568.

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