Reprinted from the Proceedings of the NATWEAL ACADEMY OF SCIEACES Vol. 46. No. 1, pp. 57-64. January. 1960. THE EFFECT OF ACRIDINE DYES ON MATING TYPE FACTORS IN ESCHERICHIA COLPt BY YUKINOHI HIROTA DEPARTMENT OF GENETICS, STANFORD UNIVERSITY MEDICAL CENTER, PALO ALTO, CALIFORNIA Conzmmicated by Joshua Lederbery, November SO, 195.9 111 &;scherichia culi, female strains are designated as F-; male st,rains are of two kinds, designated Ff aud Hfr, respectively. The male determinant in Hfr strains behaves as a chromosomal,factor allelic to F-. L--3 In F + strains, however, maleness is determined by a facto1 "F," with remarkable properties, notably it,s easy, con- tagious transmission to F cell~.~-~ Cells carrying F can be disinfected by treat- ments with cobalt iou a11d with acridine dyes.7, 8 These properties support the con- clusion that, F is a plasmid, au extrachromosomal particle, which is readily trans- ferred during mat*ing contact,s. It has been suggested that Hfr strains represent the iucorporation of F as an element of the chromosome. `, ?, 4* 5* g Jacob and Woll- man iutroduced the trr~ll "episome" for a plasmid that has a facultative association with the chronlosonle.g The present paper reports further evidence for this con- ception, name1.y ou the mechanism by which F is eliminated by the acridine dyes. Materials and :l!efhuds. ---Two acridine dyes, proflavine \ (PF, 2 : %diamino- acridine). alid acridine omnge (-10, 2 : 84isDimethylaminoacridine) were used. Stock solutions cont'ainiug 100 pg per ml of PF or 500 rg per ml of A0 in water were autoclaved atld st,ored iti t,he dark for periods up to a week. EM-sugar agarl' wa.s used as a selective medium. To grow on this medium, a recombinant must be prototrophic and also he able to ferment the sugar, e.g., lac- t#ose. Sutrient medium used for acridine treatment consisted of Difco peptone, 10 and Difco meat extract, IO gni per liter. The pH of t,he medium was adjusted with sodium hydroxide solution using t,hr Beckman pH meter. Difco penassay broth (Antibiotic assay medium number 3) was used routinely for bacteriological work. Straius of 1<. coli used in these experiments are mutants derived from strain K-12. The production, origin, and charaetcrint'ics of t,heac mutants are summarized in Table I. The strain used for acridine treatment was mainly Wci. Recombination technique: Overnight cultures of tester strains are streaked 011 EM-sugar medium and they are cross-brushed against one loopful of the culture being tested. Kecombinants arise at. the junct,ion of the hwo cultures only in com- patihle combinations.* Acridine method: An overnight F+ culture is diluted to IO4 cells ner ml in a 58 GEN,!cz'zcsS: Y. HZRO'f.4 PROC. N. .4. s. TABLE 1 A SUMMARY OF THE STRAINS USED Strain Number Genotype Original Strain Hefrrencc W6 F+Pll- K-12 4 W 1895 Hfr,M - W6 Type Hfrs w2979 F -Mal, -Xyl? -Gal2 -Am? -- J-11 (supplied by Dr. ~~avnlli)' W3133 F-Lx - Ann Cook'O W4164 F +Lac - W31'33 Obtained by F infection w4171 F+M- Wl8~5 Spontaneous reversion (given by Dr. $. Piovick) W'!399 PM- W1895 F-refractoryIs (selected in soft agar); I6 Low fertility X F- and does not infect F- to produce F+ nutrient brot,h (pH 7.6) containing 20 micrograms per ml of acridine orange (ab- breviated AO-20) and incubated overnight at 37oC. The use of acriflavine for the removal of F has been 7------ --7--- ,0-t -x-J briefly reported in an earlier publication." AC) 0 50 CONCENTRATION OF DYES [a/ml) .L 100 has proved to be more use- ful than acriflavine, pro- flavine, or acridine yellow by virtue of its low toxic- ity . The F-- clones obtained in these experiments have been repeatedly &died. The F- strains so obtained are genetically stable for t.his mating type, like the other F- report,ed. If an! Ff cells had reappeared in the F- culture by spon- taneous mutation, the> would be amplified b> c~ros+infect.ion during rc- FIG. l.--Effect of pH on the elective concentration of acridines. (a) Pro&vine, pH 7.65. (b) Proflavine, pH peated passages. 7.20. (c) Acridine orange, pH 7.60. A&dine orange, pH Experimental Results. 7.20. An overnight culture of W6 was inoculated into broth (107 cells/ml, pH 7.20 or pH 7.6) containing the indicated dye 1. Enr!ironmen&l qlfrrts and incubated at 37" C for 20 hours before plating. on the actim of acridines: The conversion of SCX- compatibility after growth in acridine broth was observed by a standard test, of sex-compatibility on isolated clones. The results are generally expressed in terms of the conversion fraction, i.e., the proportion of clones, after treatment which have become stably F-. The pI1 influences the effect of the dyes; for ex- ample, AU-50 gives 100 per cent, F- at, pH 7.6 but none at pH 7.2. Generally speaking, the minimum effective concentrat,ion of acridinc is low at a high pH and vice versa (Fig. 1). All t,he colonies formed in the untreated control remained Ft. Proflavine exerts its action at lower concentrations than that of A0 at, the same pH. However, the rate of conversion ix decreased at higher (bacteriostatic) cow centrations of PF. These pH effects indicate t,hat either the drug cation or all VOL. 46, IRA0 GENETICS: Y. HXROTA 01 I I I I I I 100 10' to2 103 104 105 IO6 IO7 i BACTERIA INOCULATED FIG. 2.-Effect of inoculum size on conversion of sex-compatibility. \`arlous dilutions of an overnight culture of W6 were inoculated into broth containing A0 (pl-I 7.6, 20 pg./ml) and incubated overnight at 37oC. anionic bacterial receptor is effective in the elimination of F. The same relationship between bacteriocidal action of acridine ion and pH effect has been extensively studied by Albert. I2 The inoculum size of F+ cells also influences the rate of cow version, which decreases with larger inocula (Fig. 2). With an inoculum of about, 1 cell per ml, 100 per cent F- can be obtained in nutrient broth at pH 7.6 with A0 10 t,o AO-20. The lowest, concent,ration at which any effect was noted was ho-1 to AO-5. The rate of elimination is reduced at lower temperatures and was essentially zero at 5'C (Table 2j. The viability of the cells obtained from the t'reated culture and the ullt'raatJed cult,ure at low t,emperature is the same. TABLE 2 EFFECT OF TEMPFXL~TIJRE OS CONVERSION OF SEX-Co MP.\TIBILITY FROM I;+ TO F- --Treated with AO-- 7Untrrated Control-. Temperature ("C): Total colonies tested: F- obtained: Per cent of F-: 37 .i 110 110 ii.7 34.6 37 5 110 110 Z.0 2.7 3 10; cells per ml of an overnight culture of W6 w-BTP inoculated into nutrient broth pH 7.ti and treated for about 20 hours with 50 microprams of acridine orange, or without it as control. Figure 3 shows the conversion fraction in treated cultures plated on EMB lactose agar at various times. F- clones first appear after a lag of 1 to 2 hours, and their incidence then increases ra.pidly. The concenkation of AO, pH of the medium, and cultural age of the t,reated cells influence the rate of conversion and the time for removal of F. Acridines are ineffective on cells growing in a synthet,ic medium.13 However, peptone 0.02 per cent,, restored t'he effect (Table 3). h systematic analysis of 60 GENETICS: Y. HIROTA ' hoc. N. A. S. -dyj -------_ 0 --------------------y. 0 60 120 180 240 TIME OF TREATMENT (minutes) FIG. 3.-Time analysis of acridine treatment. An exponential1 growing, W6 culture was inoculated into penassay broth (pH 7.2) wit K or without A0 (36 rg./ml). The F+ cultures (10' cells/ml), were shaken gently at 37oC. Samples were diluted and plated on EMB agar. The following morning single colonies were tested for their com- patability. The figure summarizes five experiments. About 2,ooO colonies from untreated controls were all F+. likely constituents of complex media showed the necessity for the simultaneous presence of several co-factors: amino acids plus a nuclein base. A0 eliminated F from F+ bacteria in a defined medium containing the following supplements: serine, aspartic acid, isoleucine, valine, and cytosine. The significance of these co-factors for the cure of maleness remains for future studies. The specific supplements re- quired for growth of auxotrophic mutants were also essential for F elimination. TABLE 3 CO-FACTOR FOR REMOVAL OF F BY A0 Minimal plus Indicated Supplemknt ,---PPT Cent of F- Obtained-- wti (M -) W4164 (Prototroph) No supplement 0 0 Peptone* plus A0 100 Mix~~ret `minus A0 71 100' 0 0 `L minus serine 4.1 10.1 `I " cytosine 3.7 c <` `, methionine 0 - 3z.4 * 200 micrograms per ml. t Concentration of each supplement: nine 20, A0 7.5 (micrograms/ml). cytosine 100, swine 20, aspartic acid 20, isoleucinr: 20, saline 20, methio- Overnight cultures of W6 and W4164 were washed with sterilized water, treated in several media, purified on EMB agar. and their compatibility tested by the cross-brushing method. The experimental conditions yier'e a8 follows:inoculum sizeabaut 104 cells per ml, t,ime of treatment 20 to 24 hours at 37'C. Methylene blue and thionine which are structurally analogous (Fig. 4) to X0 antagonize the elimination of F but not the inhibit,ion of growth by AO. These dyes alone have no dramatic activity on F- disinfection; F- clones have appeared in- frequently in treatments with methylene blue. L41bert12 has summarized analogous examples of "therapeutic int'erference" in the typanoaidal act,ion of these dyes. VOL. 46, 1960 GENETICS: Y. HIROTA (il Acridine oranqe Methylene blue Proflavine Thionine FIG. 4-Comparison of the chemical structure of several dyes. Propamidine isethionate (Baker and May Ltd., 4 : 4'-diamidino-diphenoxypropane di-(&hydroxyethane sulfonate)) is another chemotherapeutic agent which sup- posedly reacts with nucleic acids. At 50 micrograms per ml in penassay broth it was found to produce about 15 per cent F- after overnight treatment, and.is there- fore a weak agent affecting F. It is concluded that acridine mainly produces its effect on actively multiplying cells: in the absence of cell multiplication, i.e., with large inocula, low temperature, nutritional limitation, or bacteriostasis by excess dye, there is little or no loss of F. 2. Mode of action of acrid&e: The increase in the proportion of F- after acridine treatment could be attributed in principle either to mutagenic effects of acridine dyes or selective enrichment of F- cells already present among the F+ population. No differential in growth of F+ vs. F- cells could be found even remotely approach- ing the dramatic differences needed to account for complete conversions (see Table 4). However, the most decisive experiments concerned the fate of small numbers of cells inoculabed in AO medium. TABLE 4 RECONSTRUCTION EWI~W.C~NT ----- Input Mixtures . -----Mixed Cultures after Incubation---. Lac+ F- Lar + F + Lac- F- Lac- li b Lac+ F- 1.w + F + I.ac-F- Lac- F+ A Treatment 0.329 0 0 0.671 0.471 0 0.529 0 (W2979) (W4164) (W2979) F-elimi- nation Untreated 0.363 0 0 0.637 0.246 0.152 0 0.602 Control (W2979) (W4164) (W2979) F-infec- (W4164) tion Overnight cultures of W4164 (F +) and W2979 (F -) were mixed and about 101 cells/m Iinoculated into broth pH 7.6, with or without AO. and incubated overnight at 37oC. The proportion of Lac + and Lac- bacteria in the m&d cultures WP.S diagnosed on EMB lactose agar before and after the treatments; the F status of samples of inolated colonies wa8 tested by crosses against a standard F- strain (see Materials and Methods). show th8 fraction of colonies in each class. The figures in this table The mutagenic effect of A0 was tested more directly by cultivating one or two cells in a microdroplet of penassay broth, with or without acridine dye, in an oil- chamber as described by Lederberg.14 The microculturcs were then allowed to grow for several generations. Each microclone was transferred to EMB lactose agar by a capillary pipette and plated out to give single colonies arising from in- dividual cells. The colonies were then tested for sex-compatibility. The entire clone was plated; thus, differential growth of spontaneous F- mutants could alter the proportion of F- progeny within a clone but should not affect the number of 62 `lJAHT,E 5 INDUCTKIN OF F-- FROM SIXGLY ISOLATED F+ CELL ASD F+ CELLS Number of Cells Number of Drops in Drop Initially with Induced F- pridine-treated &l(s) 3* 25 2 5t 20 Untreated Contsral 1 0 a5 2 0 30 An exponential culture of strain W6 was yrown at Xi'C in penassay broth with gentle shaking. This culture contained 109 cells per ml. Single cells wew then isolated in micro- droplets by R simple modification of De Fonbrnne's oil chamber method.`a Droplets were dis- pensed by free band manipulation from a suspension containing 106 cells prr ml. with or without AO-40. The droplets were then examined microscopically. and those which were rcnfied to contain precisely one cell or two celL then recorded. They were Incubated at 37OC for 4 lmrlrs. At the end of this t,ime, the cells that, had grown in each drop were counted. then collected by a capillary pipette and spread on EMB agar. All the colonies formed on the agar were tested for sex-compatibility. If one or more F- colonies IX-PPF ohserrcrl, this was recorded as "containing induced F-." By Fisher's exact test, P = 0.0056. Total numbers of F- cells in "induced drot~" WP~P: * l,l,l. t 20.2,4,1.2. and the total cell numbers of thoseinduccd drol)trt,n wcw 43,20,10; 72,10.34,11.71, respcctiwty. clones in which F-- progency are found. As shown in Table 5, A0 increases the proportion of F--cont~aining clones from O/45 in controls to 8/G in t,he treated series. This supports the conclusions that X0 induces the loss of F from growing F+ cells. 3. The action of A0 on various rnalc strains: A wide variety of F+ strains I~U- tants of E. coli K-12 have been tested. The markers such as nutritional require- ments, drug resist,ancq lysogenicity, phage re&ance, sugar ferment&ion seem to have no bearing on t.he effect of A0 nor are they altered by the treatment. How- ever, Hfr males differ markedly from F+. For example. Table 6 shows a result of TABI& ti acridine treat,metlt on four male types all derived from WG. Mutants which show chromosoma,l segregation and non-infectivity of t)hc male chara&r are not, sensitive to the disinfecting action of acridine dyes. This illaccrAbility is observed both in stable Hfr and the F' mutants (summarized t)y Lcdcrberg alld Lederberg).`> Hirota and Iijimas reported that A0 gave F-- types not 0111y from F+ hut also from Hfr*.fi However, t,his culture has proven to bc estremcly mutable and spontn- neously produces many F+ reversions. The F-- obt)ained from cultures labelled Hfr:! can be accounted for by their origin from such F-- reversions. The F+ state cJf t>he mutable Hfr,? is also accessible to this treatment'. Stable Hfr strains? e.g., Hfr, have not been influenced by exposure t.o AO. On the other ha,nd, the other VOL. 46. lY60 GENETICS: Y. HIROTA 63 mutants which maint,ain a contagious F are readily disinfectable by this treatment. F elimination by A0 thus differs from the correlated select,ion of highly motile F- bacteria in soft, agar, which selects F- not only from F+- but also from stable Hfr 5t,rsins.`6 Discussion. -In this phenomet1on, many hypot,heses may be considered. From the above experiment, however, it, seems difficult to escape the conclusion t,hat induc- tion of F- from Ff is the result of the loss of F particles endowed with genetic continuity. Whether the relative multiplication of F is decreased, or the F particles are agglutinated by combination with t,he acridinium cation, growing cells exposed to A0 eventually produce bact,eria without F. St,ability of F- means a permanent loss of I?. and conversely t,he stable main- tenance of the F+ traits and the remarkable infectivity of F in usual media signify t,hat F mult,iplies faster than the host, cell. The transfer of F unlinked t.o the host chromosome, and t,he non-segregational uniform inherit,ance of F in the cross F+ X F-,4-6 would nl(Jst logically correspond to cytzoplasmic t~ransfer of the male det,cr- minant,.l7* `8 That is, the factor in an F+ cell is a plasmid.8~ I71 `8 Other plasmids, such as the respiratory factors in yeast, kinetoplasts in t.rypanosome are also known to be accessible to acridine treatment. 1g+2 On t,he ot,her ha,nd, malejless in Hfr cells is inherited chromosomally as a gene linked t.o certain markers in gcuet,ic recombination t&s.`+" Correlated with this is the fact that Hfr males are resistant while infective I?+ males are accessible to the acridine treatment (Table 6). These fact>s suggest that in Hfr strains this male determinant is bomld to the particular chromosome site, and thus accounts for the non-infect,ivity and segregat,ional inherit,ancc of malenrs:p! alld its inaacessihilit,y to t.he disinfect,ing a&ion of acridine dyes. This Atuation para,llels the lysogenic system of bacteria ill which there are two states, \-eget,ative phage, free in the cytoplasm and prophage> bound t,o the chromo- yome. The term episome has \)cken used for thi:: type of part,icle.g Acridille dyes are reported to curt virus infection, produce defective virus, and induce virus mut,ants respectively.23-27 However, they do not, affect, the chromo- somally bound prophage of temperate phages."* The detailed mechanism of action is, however, not known. These dyes do possess the unique property of straining ccrtSain nucleic ac,id-~ollt,ainiug particles, nuclei, and the other particles of living cells, in several organisms. 3g NurlrAdes, nucleic acid, and polyadenylic acid form complex salts with ncridinc dyes. Sol 3' The effect of acridine orange on the function of I)XA as a primer for it,s enzymatic replication has not been studied. The co-fact.ors required for F-disinfection may act in part by supporting the growth of t'he hobt, cell in its particulate components in the presence of AO. The F parMe itpelf ma,v have a tregative charge, so t,hat, acridine exerts its effect only as a (taGon. Su~~mr~.- Acridine orange converts I I'+- (male) clones of E. coli into stable F- [female) forms. The main factors which affect the rate of conversion of F+ to Fp are t,he concentration of acriditlium ions in the treating medium and the growth of t,he t'reatcd cells in the presence of the dye. Furthermore, the accessihilit,y of F tSo acridine treatment depends upon the state of F in the host cell. Xcridine orange increases the frequency of conversion from Ff to F- direct.13 without appreciable selective growth. The conversion from F+ to F-is irreversible, 64 GENETICS: Y. HIROTA PROC. N. A. S. as expected for the loss of a genetic particle. Hfr males are resistant, to the disinfect- ing action of acridine dyes. These results are well accounted for by the dual nautre of F, chromosomal F, and pla.smid F. I wish to express my heartiest thanks for the guidance given by Professor Joshua Lederberg in this study at the University of Wisconsin. I am also indebted to Dr. Esther M. Lederberg for her helpful criticism of the manuscript. I also wish to thank Professor Hideo Kikkawa of the University of Osaka for his advice and encouragement during the course of this study. * This work has been supported by research grants from the National Cancer Institute of the U.S. Public Health Service and from the National Science Foundation. t This study was done in partial fulfillment of t,he requirements for the degree of Doctor of Science at the University of Osaka. 1 Cavalli-Sf x-za, L. L., and J. L. Jinks, J. Genet., 54,87 (1956). p Richter, A., M.S. thesis, University of Wisconsin, Madison, Wise. (1957). 3 Wollman, E. L., F. Jacob, and W. Hayes, Cold Spring Ha&. Symp. f&ant. 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I,., Virology, 4,53 (1957). *$ Armstrong, J. A., Exptl. Cell ZZesearch, 11,640 (1956). ,XJ Morthland, F. W., I'. P. H. De Bruyn, and N. H. Smith, ET@/. (`ell Rewwch, 7, 201 ( 1054). 3' Beers, R. F., D. I>. Hendley, and R. F. Steiner, Nature, 182, 212 (1958).