effect of folic acid analogues on growth and cell … · effect of folic acid analogues onmicrobial...

EFFECT OF FOLIC ACID ANALOGUES ON GROWTH AND CELL DIVISIONOF NONEXACTING MICROORGANISMS'

WALTER J. NICKERSON AND MICHAEL WEBB2

Institute of Microbiology, Rutgers University, New Brunswick, New Jelsey

Received for publication June 6, 1955

Studies on the action of analogues of folic acidon microbial growth have been limited mainly tospecies which require an exogenous supply offolic acid or its derivatives for multiplication.They have been designed principally to deter-mine the nature of the relationship between in-hibitor and normal metabolite. The pertinentliterature may be found in recent reviews (Mar-tin, 1951; Petering, 1952). The effect of analoguesof folic acid on microorganisms which do notexhibit a requirement for exogenous folic acidhas been studied less frequently. Such formsusually appear to be susceptible to the analogues,but in relatively higher concentrations (Franklinet al., 1949; Nimmo-Smith and Brown, 1953).The changes induced in susceptible microorgan-isms by the analogues of folic acid have notbeen investigated in detail, although there ispresumptive evidence from experiments witlhhigher forms that such changes are analogousto those which result from a deficiency of thenormal metabolite.

In addition to the many dependent metabolicprocesses, folic acid, or the coenzyme form offolic acid, appears essential for mitosis in avianand mammalian cells. Recent work by Jacobson(1954a, 1954b) has shown that the addition ofaminopterin to osteoblasts or fibroblasts in tis-sue culture causes a specific arrest of mitosis atmetaphase. This inhibition can be counteractedby the citrovorum factor (leucovorin) but notby folic acid. It appeared of interest, therefore,to (letermine whether analogues of folic acid werecapable also of inhibiting cell division in culturesof microoiganisms, particularly those which do

1 Supported in part by a grant from the NationalInstitutes of Health, United States Public HealthService.

2 Special Fellow of the National Institute ofMicrobiology, National Institutes of Health, Be-thesda, and Visiting Fellow in Microbiology, Rut-gers University. On leave of absence from theStrangeways Research Laboratory, Cambridge,Englan(l.

not exhibit a nutritional requirement for folicacid. Tunnicliff (1939) reported that the inhibi-tion of cellular division produced in bacterialcultures by sulfonamides may be readily observedwith organisms of simple nutritional require-ments. As is well known, miciobial growth (in-crease of cellular substance, or volume) and celldivision may be considered to some extent asseparate and independent processes. In someorganisms growth in the absence of cell division,with the formation of filamentous cells, may beinduced readily by a variety of factors. How-ever, apart from the observations of Nickersonand Mankowski (1953) that aminopterin (5 X10-4M) induces an extensive formation of filamen-tous cells in a medium which normally supportsthe yeast-type of growth of Candida albicans,no description has been given of the ability ofthe folic acid analogues to inhibit cell divisionselectively in microorganisms.

MATERIALS AND METHODS

Reagents. Folic acid (pteroylglutamic acid),leucovorin, and the folic acid analogues: aminop-terin (AM)/\), 9: 10-dimethylfolic acid (DMF),9-methylfolic acid (MFA), methylaminopterin(MA\I\1), dichloroaminopterin (DCAM), andamino-an-fol (AAF) were kindly supplied bythe Calco Division of the American CyanamidCompany. Concentrated solutions (10-2 or 10-3M) of the vitamins oIr the analogues in the ap-propriate medium were prepared before eachexperiment and sterilized by filtration throughsintered glass funnels (U.F. porosity). Aliquotsof these solutions were then suitably dilutedwith the basal media. No significant decrease inconcentration occurred through adsorption ofthe compounds on the filters during sterilization.For the gradient plate method of Bryson andSzvbalski (1952), concentrated solutions of thecompounds were added to the agar just beforesolidification. Every effort was made to protectthe media from light; cultures were incubated in

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darkness to minimize photochemical degradationof the folic acid derivatives and analogues.Organisms studied. From exploratory tests in

which the growth-inhibitory activities of theanalogues were determined for a variety of or-ganisms, there were selected for further studyyeasts and bacteria which were resistant (Sac-charomyces cerevisiae; Pseudomonas fluorescensstrain 68) or particularly susceptible (Candidatropicalis, Escherichia coli strain B; Bacilluscereus strain 8; Bacillus megaterium strain 10)to the action of the antagonists. These initialexperiments, inivolving several species of grampositive and gram negative bacteria, also revealedthat there was no relationship between the sus-ceptibility of bacteria to analogues of folic acidand their gram-staining reaction.

Culture media. The effects of the analogueswere determined wherever possible in simple,chemically defined nutrient solutions, since theinhibitory activities on both growth and celldivision were markedly influenced by the com-position of the culture medium. In a 2 per centpeptone-water medium, for example, the bac-teriostatic activities of aminopterin and amino-an-fol against E. coli were only 25 per cent ofthose in the defined medium. In the paper byWebb and Nickerson (1956), certain amino acidsare shown partially to overcome the inhibitoryeffects of the antagonists.

For studies with yeasts, culture media consistedof a basal solution: KH2PO4, 3.0 g; (NH4)2S041,3.0 g; CaCl2, 0.25 g; MgSO.v 7H20, 0.25 g;dextrose, 20 g; distilled water, 1 L; and sup-plemented with yeast extract (0.2 g/L) andasparagine (2.5 g/L) for S. cerevisiae, or withbiotin (5 ,ug/L) for C. tropicalis. A modificationof Heigener's (1935) medium, in which the con-centration of each amino acid was reduced by50 per cent, was employed for cultures of B.cereus, and also for the comparative bacterio-static tests with gram positive and gram nega-tive organisms referred to above. B. megateriumwas grown in the simple nutrient solution de-scribed by Knight and Proom (1950). Monod's(1942) chemically defined medium was used forstudies with E. coli and certain other gramnegative bacteria (Serratia marcescens, A.T.C.C.strain 990; Aerobacter aerogenes; P. flutorescens).

Conditions of incubation. Yeasts were grownat 28 C in 250 ml Erlenmeyer flasks, each con-taining 50 ml of medium, on a rotatory shaking

machine. Bacterial cultures were grown undersimilar conditions and also in test tubes, eachcontaining 5 ml of medium, which were mountedon an electrically driven turntable inclined atan angle of 30 to 350 to the horizontal. Theflasks and tubes were inoculated respectivelywith 0.20- and 0.02-ml amounts of a uniformsuspension of the appropriate organisms fromcultures in the early stationary phase, previouslyequilibrated with the defined medium by serialtransfer. Such inocula for a given organism werereasonably uniform, since the maximum growth(determined turbidimetrically) reached in controlcultures cultivated with mechanical agitationunder the above conditions was reproducible.In the bacteriostatic tests, no significant varia-tions in the activities of aminopterin and amino-an-fol against E. coli were observed when stand-ardized inocula were derived from parent culturesin the lag, log, or stationary phases of growth.

Measurements of growth. Growth was measuredturbidimetrically with either a Lumetron orKlett colorimeter (filter nos. 420 and 47, respec-tively), either directly on the growing culturesin test tubes or on suitably diluted aliquotsremoved aseptically at convenient intervals fromthe shaken flasks. DIry weight determinationswere also carried out at the end of experimentsin flask cultures. WVith the yeasts, aliquots ofthe cultures were fittered through sintered glasscrucibles (porosity F), the cells washed thor-oughly with water and then dried to constantweight at 80 C. Bacterial cultures were centri-fuged in tared 15-ml conical centrifuge tubes,the cells washed three times by alternately re-suspending in water and recentrifuging, and thetubes and cell deposits then dried to constantweight at 80 to 90 C.

Cytological methods. For cytological studies ofthe effects of the folic acid antagonists, smearsof cells were fixed in osmium tetroxide vapor,hydrolyzed, and stained according to the meth-ods described by Robinow (1944) and Smith(1950) to reveal the nuclear elements. Rob-inow's (1945) tannic acid-crystal violet proce-dure was used to stain the bacterial cell walland to demonstrate the formation of transversecell walls.

RESULTS

Effects of folic acid analogues on growth anddivision of certain yeasts. S. cerevisiae proved

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completely resistant to the action of all of thefolic acid analogues examined. No inhibition ofgrowth (table 1) was detected in liquid culturescontaining AM, DCAM, DMF, MFA, MAM,or AAF over a range of concentrations. The addi-tion of aminopterin (10-4 M) to cultures earlyin the exponential phase of growth had no effecton the form of the growth curve. In fact, at thisconcentration aminopterin appeared to stimulategrowth slightly in comparison with the controlcultures. This, however, could not be attributedto deamination of AM to yield folic acid, sincefolic acid (10-4 to 10-6 M) failed to increaseeither the rate of growth oIr the final crop yield.Examination of cultures of S. cerevisiae on

gradient plates containing the various com-

pounds failed to reveal any visible inhibition ofgrowth with increasing concentrations of theanalogues. On prolonged incubation of platescontaining aminopterin and dichloroaminopterin,the cells accumulated a yellow pigment; thiswas particularly marked at concentrations ofaminopterin greater than 1.2 X 10-4 M and ofdichloroaminopterin above 5 X 10-4 M.

In liquid cultures particularly, C. tropicalisproved more susceptible to folic acid analoguesthan was S. cerevisiae, although after 72 hours'incubation, cultures usually recovered from theeffects of the analogues (other than dichloro-aminopterin, table 1). Cells from cultures whichhad recovered from the effects of aminopterinwere usually colored bright yellow, indicatingan accumulation of the compound, or its degrada-tion products, during the period of recovery.

This colored material was not removed fromthe cells by washing with water or 0.1 M buffersolutions (pH 5 to 7). It appeared to be locatedintracellularly, although it was not possible toestablish its association with any particular cellu-lar structure by the examination of dilute sus-

pensions of the cells under the fluoresecentmicroscope.When incorporated into gradient plates the

growth inhibitory effects of the active analogueswere much less pronounced than in liquid media.After prolonged incubation (7 days at 28 C)streaks of C. tropicalis were surrounded at thelower concentrations (10-5 to 10-6 M) of theanalogues (DCAM, AM, MAMI, DMIF, MIFA,

TABLE 1Effect of folic acid analogues on growth of yeasts

Ageof Molar Concentration of AnalogueCultures, Hrs. Organism and Analogue

0 10-6 10-5 10-4

Dry weight measurement of growth (mg/100 ml culture)

S. cerevisiae48 Methylamiinopterin 532 _ 525 54048 Aminopterin 545 - 545 54372 Aminopterin 785 771 792 814

C. tropicalis24 Dimethylfolic acid 144.5 149.5 139.5 156.548 Dichloroaminopterin 237.2 252.0 38.6 13.572 Aminopterin 230.0 230.0 200.0 184.5

Turbidimetric measurement of growth (optical density*)

C. tropicalis18 Aminopterin 0.066 0.052 0.028 0.00048 Aminopterin 0.240 0.232 -

72 Aminopterin 0.260 0.244 0.192 0.18684 Aminopterin 0.264 0.266 0.258 0.26024 Dichloroaminopterin 0.158 0.074 0.028 0.02648 Dichloroaminopterin 0.252 0.296 0.050 0.03024 Amino-an-fol 0.156 - 0.150 0.15048 Amino-an-fol 0.260 0 .258 0.25424 Dimethylfolic acid 0.150 0.150 0.140 r 0.156

* Cultures diluted 1:5 before reading made.

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c') 0zIi

< 0.8U

0.6 -

0.4-

0.2-

240 280 320 360 400WAVELENGTH mr

Figure 1. Ultraviolet absorption spectrum ofbutanol-soluble yellow pigment accumulated byCandida tropicalis during growth on agar platescontaining 5 X 10-5 M to 10--' Mi aminopterin.

and AAF) by zones of filamentous outgrowthindicative of the Y MI conversion, alreadyextensively investigated (Nickerson, 1948; Nick-erson and Van Rij, 1949). In contrast to previousfindings on the development of filamentous cellsin aging cultures of Candida (Langeron andGuerra, 1939; Nickerson and Chung, 1954),these zones were observe(d particulaily betweenthe parallel streaks iather than at their ends,or around the peripheral margins.The presence of most of the folic acid ana-

logues studied caused markied alterations in themorphology of C. tropicalis grown in liquid cul-tures. Dumbbell-shaped cells were present as wellas many long forms (figure 3) consisting of astring of cells formed by terminal budding with-out abscission. SuchI foirms were particularlycharacteristic of C. tropicalis during the first 12to 18 hours of gr-owth in the presence of 10-5 Mdichloroaminopterin (table 1).On gradient plates containing aminopterin at

concentrations between 10-5 M and 5 X 10-5 M,

there was a iapid change in color of the growthalong the streak (from cream to yellow); at con-centrations above 5 X 10-5 M, C. tropicalis ap-peaied bright yellow. At concentrations in excessof this value, small, intensely yellow secondarycolonies developed on the primary growth duringthe extended period of incubation. Such coloniescontained a high proportion of filamentous cellsand, when transferred to the liquid mediumwithout aminopterin, exhibited a prolonged lagphase but otherwise normal growth curve, andgave rise to a population of unpigmented cellsof unaltered susceptibility to the antagonist.

Extraction of a buttanol-soluble pteridine fromC. tropicalis cells grown with aminopterin. Muchof the yellow color was removed from the pig-mented yeast cells (harvested from agar platescontaining aminopterin at 5 X 10-5 to 10-4 M)by extracting washed, aqueous suspensions ofcells with redistilled n-butanol for 24 hr in thedark at rioom temperature. The combined butanolextracts, when washed with water (3 times) andevaporated to dryness in vacuo, yielded a paleyellow solid. No such pigment wvas obtained byrepetition of the above procedure with cellsgrown on agar in the absence of the analogue,or with the uninoculated, aminopterin-contain-ing agar.The ultiaviolet absorption of the extract from

the colored C. tropicalis cells over the range 200to 400 m,4 is shown in figure 1 (coirrected forpresence of butanol soluble materials from coIn-trol cells and uninoculated medium). The foImof this curve is suggestive of a free pteriidine.The presence of a free pteridine in the yeast cellsis also suppoited by the solubility of the materialin n-butanol, which suggests that the pteridinearises by reductive or hydrolytic, rather thanby oxidative cleavage of the -CH2 NH-

cleavage

o to 19HOOC-f.No-l-7"fN Cv.

HOOCX41diazotizable amine free pteridineFigure 2. Cleavage of aminopterin to yield free

pteridine and p-aminobenzoyl glutamic residute(diazotizable aromatic amine).

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linkage of the aminopterin molecule (figure 2).Although the latter possibility is not supportedby the evidence presented below, it cannot bedismissed entirely since exhaustive extraction ofthe yellow C. tropicalis with butanol (or otherorganic solvents) failed to remove the colorcompletely from the cells. The residual colorprobably is due to the accumulation by the cellsof the intact analogue.The nature of the pteridine residue present in

the butanol soluble fraction of aminopterin-treate(d C. tropicalis was not investigated.3 Itmay be pointed out, however, that the compari-son of the curve shown in figuie 1 with the cor-responding ultraviolet absorption curves of2-amino-4-hydroxy-6-methyl pteridine, 2-amino-4-hydroxy-pteridine, and 2-amino-4-hydroxypteridine-6-aldehyde rieveals differences in theposition of the maxima and minima, which arepossibly greater than would be expected by the re-placement of the hydroxyl at C4 of the referencecompounds by an -NH2 group.

Cleavage of aminopterin molecule by non-proliferating cells of C. tropicalis. Washed cells(230 mg dry weight), harvested from 44-hrcultures in 100 ml of medium, weere incubatedin the dark at 28 C with a 5 X 10-4 M solutionof aminopterin (11 mg) and glucose (0.1 M) in0.2 M phosphate buffer, pH 6.2. The progressiveformation of a diazotizable amine could be dein-monstrated (method of Bratton and Mlarshall,1939) and thus permit following the cleavage ofthe analogue at the -CH2-NH- linkage (fig-ure 2). The results of duplicate experiments aresummarized in table 2. Values obtained in similarstudies with C. tropicalis cultivated in a mediumcontaining 1.75 jig aminopterin/ml suggest thatcells grown in the presence of the antagonist areslightly more active in disrupting the analoguemolecule (table 2).

3Although principally composed of the pteri-dine derived from cleavage of aminopterin, theyellow pigment extracted from Candida tropicalisis probably heterogeneous in composition. Theanalogue was of high purity but still containedsome free pteridines as contaminants; the solubili-ties of the pteridines in n-butanol vary consider-ably with pH. Therefore, it is difficult to estimatethe contribution of these components to the ab-sorption spectrum of the yellow pigment. Work isin progress, using chromatographically pure ana-logues. to identify the pteridines resulting fromcleavage of several of the anialogues.

TABLE, 2

Cleavagje of aminopterin mtolecule b!y tion-pro-liferating cells of Candida tropicalis*

p-Aminobenzoyl Residues Formed

Incubation Time Normal Cells grown withcells aminopterint

Exp. I Exp. 2 Exp. I Exp. 2

hr pm/100 Aim Antinopterin AM/1lOO M.4Aninoepterin0 0 0 03 7.0 - 10.0 11.56 12.5 18.5 12.5 20.012 24.0 - 28.014 21.0 - 26.526 45.0 - 55.0 -

* Formation of diazotizable aromatic aminiogroups from aminopterin measured by methodof Bratton and Marshall (1939). Reaction system:Aminopterin (5 X 10-4 M), pH 6.2 dextrose-phos-phate buffer, 28 C, and C. tropicalis cells (4.6 mgdry weight/ml). Aliquots (4.0 ml) removedaseptically for analysis at times shown.

t Cells grown in presence of aminopterin(1.75 ,g/ml).

TABLE 3Bacteriostatic activities of folic acid analogiies

for Bacillus megateriuni *

Folic Acid Analogue

Methylaminop-terin ..........

Aminopterin ....Dimethylfolic

acid...........Amino-an-fol....Dichloraminop-

terin ..........Methylfolic

acid...........

Dilution Giving Complete Inhibition ofGrowth

22.5 hr 44 hr 66 hr

1:40,0001:40,000

1:20,0001:20,000

1:10,000

1:20,000

* Activities determined

1:40,0001:40,000

1:10,0001:20,000

1: 5,000

1:10,000

1:40,0001:40,000

1:10,0001:20,000

1: 5,000

1:10,000

in a chemically de-fined medium by serial dilution mehtod. Dilutionsrecorded are those giving complete inhibition ofgrowth after incubation at 28 C for the timesshown.

The formation of aromatic amino groups wasnot detected in growing cultures of either S.cerevisiae or C. tropicalis. This suggests that inthe growing yeast cultures the p-amino benzoyl-(glutamic acid) residues formed by the cleavage

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6Figures 3-6. Microscopic appearance of elongated cells grown in the presence of aminopterin. All

preparations stained with crystal violet. Magnification, 1250 X.Figure 3. Mycelial (M) cells from an 18-hour culture of Candida tropicalis grown at 28 C in presence

of 5 X 10-5 M aminopterin. M-cells were also characteristic of cultures containing low concentrationsof amino-an-fol, dichloroaminopterin, or methylaminopterin.

Figure 4. Filamentous cells formed by Escherichia coli at 28 C on agar plates containing 4 X 10-4M aminopterin.

Figure 5. Filamentous cells from a 36-hr culture of Escherichia coli in Monod's liquid medium con-taining 5 X 105 M aminopterin.

Figure 6. Basophilic granules distributed along the length of filamentous cells of Escherichia colitaken from 48-hr cultures grown in liquid medium containing aminopterin.of aminopterin are rapidly utilized by the cells.It is clear that the folic acid analogues do notlead to an accumulation in these cultures ofdiazotizable intermediary metabolites as, for ex-ample, 4-aminoimidazole-5-carboxamide (Wool-ley and Pringle, 1950). The possibility that thecleavage of the aminopterin molecule by C.tropicalis is followed by deamination of theliberated amine, is discounted by the fact thatno ammonia is formed when washed cells areincubated with the analogue at pH 6.2.

The inactivation of aminopterin by C. tropi-calis was also demonstrated by seeding C. trop-icalis in small, widely separated areas on agarplates of Monod's medium supplemented withbiotin (5 ,g/L) at pH 5.5 and containing amin-opterin at a concentration (10-3 M) sufficientto inhibit completely the growth of E. coli. Ifthese plates were incubated in darkness at 28 Cfor 48 to 72 hours and then inoculated with E.coli, growth of the latter occurred in circularzones surrounding the yeast colonies. In each

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of these zones, growth was most intense in theneighborhood of the central C. tropicalis colonyand decreased markedly towards the periphery.

Effects of folic acid analogues on growth anddivision of certain bacteria. FoI B. cereus a more

complex culture medium was necessairy forgrowth and, studies of the effects of folic acidanalogues were restricted to comparatively fewexperiments with aminopterin. B. cereus provedparticularly susceptible to the analogue; con-

centrations as low as 10-5 M were sufficient toinhibit growth completely during the first 48hours of incubation while, after 72 hr, the tur-bidities of these cultures were only 33 per centof those of the controls. On gradient plates after72 hr a sharp inhibition of growth was apparentat an aminopterin concentration of 4 X 10-6 M.

Smears prepared from the cells at the limit ofgrowth on these plates contained only long chainsof bacilli, which frequently extended over severalfields of the microscope. Long chains were alsocharacteristic of 48- and 72-hour cultures of B.cereus in the liquid medium containing 10-6 Ai

or 10-5 M aminopterin; however, truly filamen-tous cells were not observed.

In the chemically defined medium of Knightand Proom (1950) all the folic acid analoguesexamined exhibited growth inhibitory activityfor B. megaterium. Effective concentrations ofthe analogues leading to complete inhibition ofgrowth are recorded in table 3. In these experi-ments chains and distorted (swollen) cells, butno nonseptate filaments, were observed in stained

TABLE 4

Influence of amino-an-fol, dichloraminopterin,dimethylfolic acid, and mtiethylan'tinopterin

on the growth of Escherichia coli*

Optical Density Dry Weight (mgcls/t00 ml culture)

Folic AcidAnalogue Molar concentration of analogue

o0 0-5 10-4 0 10 10-4

Amino-an-fol. 0. 148 0.106 0.078 131 .01108.0, 36.0Dichloramin-

opterin ...... 0. 146 0. 146 0.140 130. 8 130.8 129.2Dimethylfolic

acid ........ 0 150 0.146 0.118 135.4'130. 8 124. 8Methylamin-

opterin ... 0.148 0.148 0.144 =

* Cultures incubated for 18 hr at 28 C.

TABLE 5Comlparison of growth-inhibitory activity ofanalogutes of pteroylglutamic acid (folic acid)

for three different organisms

Eschierichia Candida Streptococ-Analogue coli tro picalis cus faecalis% inhibition % inhibition inhibition

at 18 hr at 48 hr index*

Aminopterint 82 94 700Dichloroaminop-

terin .......... 3 91 1910-Methylamin-opterin ........ 3 - 1530

9, 10-Dimethyl-folic. 15 0 3.4

10-Methvlfolic. 3 - 100Amino-an-folt... 75 3.4 -

* An arbitrary value of 100 is assigned to 10-methylfolic acid for 50 per cent inhibition ofgrowth of S. faecalis R (exacting for folic acid).Data compiled from Seeger et al. (1949), Hult-quist et al. (1949), and Cosulich and Smith (1948).

t 4-amino pteroylglutamic acid (aminopterin).I 4-amino pteroylaspartic acid (amino-an-fol).

smears prepared from cultures containing sub-bacteriostatic concentrations of the analogues.On gradient plates containing aminopterin,

growth of E. coli was sharply but not completelyinhibited at an analogue concentration of 4 X10-4 M. At this concentration 90 per cent of thecells were in the form of long filaments (figure 4)frequently about 50 to 100 ,u in length, but ofdiameter similar to that of the normal E. coli.At 10-4 M AM the incidence of these filaments wasreduced to about 50 per cent, while at a concen-tration of 10-5 M the cells were almost entirely(about 90 per cent) of normal morphology. Thegrowth of E. coli in Monod's medium was com-pletely suppressed by aminopterin at a concen-tration of 2.5 X 104 M, and partially inhibitedat concentrations in excess of 6 X 10-5 M, duringthe first 19 to 24 hours of incubation. After 48hours, however, slight growth was usually de-tected at concentrations of AM as high as 5 X10-4 M. The bacteriostatic activity of aminopterinunder the conditions of these experiments, inwhich growth was measured qualitatively, theappearance of visible turbidity after a relativelyprolonged incubation was not significantly in-fluenced by the age (2 to 24 hr) of the inoculum.The incidence of filamentous cells (figure 5)generally appeared to reach a maximum at inter-

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NICKERSON ANDWE'1[31

mediate (about 10-5 M) iathei than at the highestconcentrations of AM.

Amino-an-fol, although less effective thanaminopterin, resembled the latter in its actionon E. coli, whereas other analogues (DCAMI,AIFA, DFAIA, and AIAM. table 4) failed topioduce complete inhibition of growth over therange of concentration tested (up to 10- mI).The bacteriostatic activitv of AAI against E.coli was not appreciably affected by the presence

%

I90

Figures 7-9. Distribution of basophilic granulesin cells of Escherichia coli taken from 48-hr cul-tures containing aminopterin. Crystal violet stain,1250 X.

Figures 7-8. Granules condensed towards thecenter of the cells.

Figuire 9. Basophilic areas associated with "plas-modesmids. "

of 1: 5000 concentirations of MNIAM or M1FA;howevei, the activity of amino-an-fol was in-creased significantly by MANI. Under the sameconditions 9:10 dimethylfolic acid appeared toenhance the activity of AMI during the first 18hr of the culture, although after 42 hr such cul-tures were indistinguishable from those contain-ing A.M\ alone. In table 5 the activities of severalof the analogues aire compared for (lifferentorganisms.Growth in the absence of cell division was

also obtaine(d in the presence of low concentra-tions of other folic acid analogues; filamentouscells were particularly characteristic of culturesgrown in MIonod's medium containing 10- Mamino-an-fol, or MXIAMI. In contrast to normal(control) cells, these filamentous forms of E. coliexhibited a poor affinity for crystal violet. Piepa-rations stained in this way appeared pale mauve,with the moire basophilic components of theprotoplasm localized in small, discrete areas.These were regularly distributed along the lengthof the filament (figure 6), or condensed towardsthe center of the cell (figures 7 and 8), or in welldefined "plasmodesmids" (figure 9).The absence of transverse cell walls from the

filamentous form of E. coli was readily apparentin preparations mordanted in 10 per cent tannicacid and stained with 0.02 per cent crystal violetaccording to Robinow's (1945) method. It wasdifficult, however, to follow the effects of thefolic acid analogues on the division of the nuclearelements by the usual cytological procedures,since filamentous cells were observed only inolder cultures. Films prepared, for example, from2- to 4-hr cultures of E. coli containing 5 X 10-5M AiNI, were devoid of filamentous cells. In thesepreparations, as well as in those of 6.5-hr cul-tures, which contained only a small percentageof filaments, the nuclear elements were readilyapparent. In oldei cultures (23 hr), in which thecells were almost entirely filamentous, the nucleatrstructures weie poorly defined. This was in con-trast to cells harvested from control cultures ofthe same age, in which the nuclear elements werereadily demonstrated after appropriate hydroly-sis. It is possible that the failure to obtain con-vincing recordls of the nuclear structures of thefilamentous cells bv (lirect methods may be ofsomie significance with regard to the mo(le ofaction of the folic acid analogues (Webb andNickerson, 1956). In order to demonstrate nu-elear elements in the filamentous forms it was

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necessary to transfer a thick suspension of thecells to the surface of a nutrient agar plate main-tained at 37 C. After 30 to 90 min (before multi-plication occurred) impression preparations weremade from blocks of the agar cut from the plateand fixed in osmium tetroxide vapor. The"young" filamentous cells obtained by this tech-nique then appeared to possess nuclear elementsregularly spaced along their length. Although thede novo formation of nuclear structures in pre-existing filaments seems unlikely (but see Jeenerand Jeener, 1952), the appearance of these cellsmay bear no resemblance to that of the initialfilaments, since the known increase in the DNAcontent of bacterial cells during the lag phase ofthe culture may render nonapparent the effectsof the antagonists.

A. aerogenes closely resembled E. coli in itssusceptibility to the inhibitory effects of AM andAAF on both growth and cell division; most ofthe observations recorded in the previous sectionapply equally well to this organism. In contrast,the folic acid analogues were without effect uponthe growth of P. fluorescens and Chromobacteriumiodinum, except at concentrations in excess of 10-3M. Even at these concentrations the limitation ofgrowth appeared unaccompanied by any inhibi-tion of cellular division, and filamentous cellswere not observed. S. marcescens, although re-sistant to the growth-inhibitory effects ofaminopterin and amino-an-fol, tended in thepresence of these analogues to form swollen,coccoidal cells as well as thin, distorted filaments(which had a poor affinity for basic dyes).

DISCUSSION

It is evident from the results presented thatlow concentrations of the folic acid analoguesmay inhibit cell division in certain microorgan-isms to a greater extent than growth. This leadsto the formation of filaments (figures 4 and 5)in bacteria and to the Y M conversion (Nick-erson and Mankowski, 1953) in yeast cultures(figure 3). The interpretation of the effects of thefolic acid analogues on the growth and cell divi-sion of susceptible yeasts and bacteria, however,is complicated by the abilities of these organismsto inactivate the antagonistic substances. In cul-tures of C. tropicalis, for example, inactivationof aminopterin appears to be achieved by reduc-tive cleavage of the linkage between C9 and N1owith the production of a free, butanol-solublepteridine and p-aminobenzoylglutamic acid, orits hydrolysis products.

The apparent inability of C. tropicalis to over-come the growth inhibitory effects of dichloro-aminopterin (table 1), an analogue in whichchlorine atoms are substituted at positions 3 and5 of the benzene ring, is to be expected, sincecleavage of the molecule of this antagonist be-tween C9 and N1o would give rise to a substitutedp-aminobenzoic acid residue, inhibitory to theutilization of the corresponding normal metabo-lite.The mechanisms by which bacteria overcome

the effects of the folic acid analogues will bedescribed in a subsequent paper. It may bementioned, however, that with certain species(but not necessarily with E. coli) the process ofinactivation appears to resemble closely thatdescribed above for C. tropicalis (figure 2), whichin itself corresponds in many respects to thedegradation of folic acid by liver homogenates(Rauen et al., 1951). Inactivation of analoguescontaining the 4-amino group or a methyl groupat positions 9 or 10, by deamination and de-methylation, respectively, as has been suggestedby Kidder et al. (1951) for Tetrahymena, andby Foley (1952), and Hutchinson and Burchenal(1952) for S. faecalis, has not been encounteredwith the organisms studied in the present work.Furthermore, the conditions of the experimentshave precluded the development of resistantstrains (Burchenal et al., 1951).

SUMMARY

The effects of six different analogues of folicacid on growth and division were studied with avariety of microorganisms nonexacting for folicacid. The inhibitory action of the analogues wasmarkedly lower in complex organic media thanin simple, chemically defined media.The lag phase with Candida tropicalis was

greatly prolonged by some of the analogues, butrecovery occurred (except from dichloroamin-opterin) on continued incubation. Recoveryfrom aminopterin results from enzymatic inac-tivation of the analogue, involving a cleavage(presumably reductive) of the molecule, and theaccumulation of butanol-soluble free pteridinewithin the yeast cells. Nonproliferating cells ofC. tropicalis cleave aminopterin with the libera-tion of a diazotizable, aromatic amino compound(p-aminobenzoyl-glutamic acid residue).

In Escherichia coli extremely long filamentouscells (50-100 ,u) were produced in 10-5 M con-

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centration of aminopterin, amino-an-fol, ormethylaminopterin. These filamentous cells hadvery little affinity for basic dyes, and nuclearelements were very poorly defined. The bearingof these findings on the mode of action of folicacid analogues is discussed.

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