MUTAGENESIS by ACRIDINE YELLOW in BACZLLUS Subtzlzsl

MUTAGENESIS BY ACRIDINE YELLOW IN BACZLLUS SUBTZLZSl

CHARLES R. STEWART2 Department of Genetics and Kennedy Laboratories for Molecular Medicine, Stanfora!University Medical School, Palo Alto, California Received October 13. 1967

ROFLAVIN, acridine yellow, and other amino and methyl derivatives of 'acridine are effective mutagens for bacteriophage (DENIARs1953; ORGELand BRENNER1961), causing the addition or deletion of nucleotides and a resulting shift in the reading frame for translation of the genetic message (CRICKet al. 1961; TERZAGHIet al. 1966). For bacteria, however, successful mutagenesis with acridine compounds is more difficult, and has been achieved only by combining the acridine treatment with irradiation by visible light (WEBBand KUBITSCHEK 1963), by addition of alkylating side chains to the acridine molecules (AMESand WHITFIELD1966), or by such high acridine concentrations as to cause rapid killing of the bacterial population (WITKIN1947; ZAMPIERIand GREENBERG 1965).This paper describes successful mutagenesis in Bacillus subtilis by acridine yellow, in the dark, at concentrations that cause no loss of viability. LERMAN(1963) suggested that acridine induced mutagenesis may be the result of unequal crossing over that occurs because the intercalated acridines cause imprecise pairing of the recombining DNA molecules. This suggestion is consistent with the observations that mutagenesis by 5-aminoacridine in yeast occurs only during meiosis (RfAGNI, VONBORSTEL and SORA1964) and that, in T4, a high proportion of prloflavin induced mutants first appear in heterozygotes (DRAKE1964). Howevler, this interpretation is called into question by the facts that increasing the recombination frequency does not increase the mutation frequency (DRAKE1964)., that inhibition of recombination does not decrease the mutation frequency (LEERMAN,quoted by STREISINGERet al. 1966), that the heterozygotes associated with proflavin induced mutants are more characteristic of terminally redundant than of internal heterozygotes ( STREISINGERet al. 1966), and that the expected ]production of complementary mutants has not been observed (DRAKE1964). If recombination were an integral part of the mechanism of acridine yellow mutagenesis, one would expect that such mutagenesis at a particular locus would be frequently associated with recombination for linked markers. Evidence has been presented that certain types of spontaneous mutagenesis occur in conjunction

This nark vas supported by United States Public Health Service Research Training grant GM-295, by National Insti- tute of Alleig>-and Infectious Diseases Research grant AI-5160, and by National Science Foundation grant G-6411. ' Present address: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York iUS(r1. ' Abbrei-iations: met, methionine; try, tryptophan; his, histidine; ilu, isoleucine-valine; gly, glycine; arg, arginine; mr. tyi-omie. thr. threonine; leu, leucine; AY, acridine yellow.

Genetics 59: 73-31 May 1968. 24 CHARLES R. STEWART with recombination of linked markers (MAGNI1963; STRIGINI1965; YOSHIKAWA 1966). This paper presents evidence that maximum levels of acridine yellow induced mutagenesis can be obtained under conditions where transformation does not take place, and further, that when acridine yellow induced mutagenesis occurs during transformation, there is no observed association between mutagenesis at a particular locus and transformation for markers linked to that locus.

MATERIALS AND METHODS

Bacillus subtilis strains: The mutant whose reversion by acridine yellow is most thoroughly studied here is met-63, which is carried in the following strains: SB-54 (met-6 try-2) SB-754 (met-6 his-2), SB-868 (met-6 ilu-6 try-2), and SB-871 (met-6 ilv-5). The met-6 marlier is linked to both ilu markers (presumably it is in the linkage group described by BARAT,ANAGNOSTOPOUL~S and SCHNEIDER1965). Each of the ilu markers gives about 50% cotransfer with met-6 in transformation by the DNAs used in these experiments. All other mutants and strains are identified in Table 3.

900

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600 i I I I 500 i I I / 400 I I I I 3oa

20c

TIME (MINUTES) FIGURE1.-Mutagenesis by acridine yellow in the dark in non-competent culture. An over- night culture of SB-868 (met-6 ilu-6 try-2) was diluted 1:lOO into C-2 and shaken for seven hours at 30". At this point (time 0) 1 ml was transferred to each of a series of tubes, 10 pg/ml acridine yellow added to half of the tubes and the incubation continued at 30". The cultures were plated at varying times after the addition of AY. 0-0 Met-6+ revertant frequency x 10' with AY. 0-1 Met-6+ revertan,t frequency x 107 without AY. .-e Viable count x lO-G/ml with AY. E-= Viable count x lP/ml without AY. MUTAGENESIS BY ACRIDINE YELLOW 25

Reagents: B. subtilis DNA was prepared by the method of MARMUR(1961). Acridine yellow was purchased from K and K Laboratories. Media C-l = SPIZIZEN'Sminimal medium (SPIZIZEN1958) supplemented with 0.5% glucose and amino acid groups I-V at 6.2 x the concentrations recommended by LEDERBERG(l%O) (except 3.1 x for histidine). C-2 = SPIZIZEN'Sminimal medium supplemented with: 0.5% glucose; 0.02% casein hydrolysate (acid hydrolyzed and neutralized to pH 7.0) ; 10 pg/ml each of histidine, phenylalanine, tryptophan, and tyrosine; and 0.2 pg/ml each of p-aminobenzoic acid and p-hydroxybenzoic acid. Transformant and revertant colonies were scored on plates containing 1.5% Difco Bacto-Agar plus 0.5% glucose in SPIZIZEN'Sminimal medium (SPIZIZEN1958). The appropriate amino acid supplements are added to a concentration of 25 pg/ml. Total viable count was scored on plates of Bacto Nutrient Agar. Preparation of competent' cells: A 5 ml solution of Penassay broth (Difco Antibiotic Medium 3) is inoculated from either a slant or a single colony and shaken at 37" for 12-13 hours, centri- fuged, and the pellet resuspended in 5 ml C-l. 0.5 ml is added 'to 5 ml C-l and shaken vigorously at 37" for 4% hours. 0.1 or 0.2 ml aliquots of this culture are then added to 0.9 mI C-2 and incubated, with slow shakin,g, at 30" for 9.0 minutes. At the end of the 90 minute period, DNA and/or acridine yellow is added and the incubation continued in the same medium. Incubation with acridine yellow: During the treatment with acridine yellow. all manipulations are performed in subdued yellow light. and the incubation itself is done in complete darkness.

RESULTS

Induction of reverse mutations by acridine yellow: Figure 1 shows the time course of mutagenesis during growth at 30" in C-2 in the dark. The revertant frequency increases with time of incubation with AY3 until, at 360 minutes, it is about 33 times the frequency without AY. There are two explanations, alternative to mutagenesis, that could conceivably account for a higher observed revertant frequency with AY than without AY. One is that the AY may be having a selective effect, permitting more rapid growth of revertants than of the bulk of the population. That this is not the case is shown by the reconstruction experiments in Figure 2. met-6- cells were mixed with met-6f revertants, which were present at more than lo3times the usual revertant frequency. Under these conditions, incubation with AY produced no increase in the frequency of revertants, showing that the AY does not confer a selective advantage on the revertants. The other alternative explanation is that competitive auxotrophic suppression (RYAN and LEDERBERG1946; GRIGG1965) may be differentially affecting the plating efficiency. This possibility was tested by a reconstruction experiment in which a pure culture of revertants was plated separately or mixed with a met-6- culture which was plated at the usual cell concentrations. As shown in Table 1, there is no significant auxotrophic suppression. Lack of association of mutagenesis with recombination: The experiment shown in Figure 1 was done with non-competent cells, as shown by the failure of aliquots, taken from the cultures at various times during incubation. to be transformed by DNA from prototrophic B. subtilis. Similar (but not higher) levels of mutagenesis with AY can be achieved with competent cells, although in this case there is usually a lag of about two hours after the addition of AY before the 26 CHARLES R. STEWART

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FIGURE2.-Lack of selective effect of acridine yellow. met-6- cells were mixed with a 100 fold lower concentration of met-64- revertants and grown at 30" in C-2 with and without 10 pg/ml AY. Samples were plated at various times after the addition of AY. 0-0 met-6f frequency x 104 with AY. 0--0 met-6f trequency x 104 without AY. 0-0 Viable count x 10-F/ml with AY. E--. Viable count x IP/ml without AY. revertant frequency begins to increase, and the revertant frequency often reaches a maximum after about 4 hours incubation with AY in C-2 at 30". The addition of DNA carrying the same marker makes no difference in the reversion frequency. The latter observation, added to the success of mutagenesis in non-competent

TABLE 1

Luck of auxotrophic suppression

Number of Mean number of Culture plated colonies per plate colonies per plate SB-754 427,520,516,541 501 met-64- revertant 505,591,684,618 600 SB-754 and met-6f revertant 1113,1192,1257,1492 1264.

Cultures of both SB-754 (his-2 met-6) and its met-6f revertant were grown through the usual competence procedure plus the four hour incubation standardly used for the mutagenesis procedure. 0.1 ml aliquots of the SB-754 culture and of a l:lO5 dilution of the met-Cf revertant culture were plated separately and together on plates lacking methionine but containing histidine. IVIUTAGENESIS BY ACRIDINE YELLOW 27 cells, suggests that recombination between exogenous and endogenous DNA is not necessary for AY induced mutagenesis. This was tested more directly by look- ing for a correlation between reversion of met-6 and recombination for markers linked to met-6. Competent SB-871 was simultaneously treated with AY and with transforming DNA from SB-54. The recipient strain is mutant for both met-6 and ilv-5. The donor DNA is mutant for met-6 and wild type for ilu. The results are shown in Table 2. There is no case in which a cell that has been reverted to met-&+has also been transformed to ilu-5 +. It should be noted that the AY is strongly inhibitory to transformation. Experi- ments on the mechanisim of this inhibition have been inconclusive. Induction of forward' mutations: Efforts were made to induce forward mutations with AY. A number of auxotrophic mutants appeared after AY treatment of SB-32 (his-2), followed by several generations of growth in non-selective medium. However, the rate of appearance of such mutants was not significantly above the rate of spontalneous mutagenesis and none of the new auxotrophs were revertible by AY. The frequency of streptomycin resistant mutants was frequently. but not always, increased to about twice the spontaneous frequency by AY treatment followed by at least two generations growth in non-selective medium. Suruey of other mutations: AY was tested for its ability to revert a variety of mutations. As Table 3 shows, about half of the spontaneous mutations tested had their reversion frequency increased by AY, while none of the mutations induced by UV. nitrous acid, or nitrosoguanidine could be reverted by AY.

DISCUSSION These experiments show that acridine yellow is mutagenic for Bacillus subtilis, and that successful mutagenesis requires neither visible light nor bactericidal levels of the dye. The observation that there are two classes of mutations, one revertible by AY and the other not, is consistent with similar observations in bacteriophage (ORGELand BRENNER1961), as is the observation of a relatively high proportion of spontaneous mutants that are revertible by acridine yellow.

TABLE 2 Absence of recombination for linkzd marker in acridine yellow induced mutants

Number of colonies met-6f colonies ilu-5' colonies ~- that are that are Viable count met-6+ ilu-5+ (met-6f ilv-5+) also &-5+ also met-6+ ______~____.___. ___~~.~~ Without AY 1228 x 105 655 319 X lo1 0 0/2m 0/200 With AY 725 x 105 4353 12 x 101 0 0/200 0/12

0.3 pg/ml SB-54 DNA (met-6 try-2), with or without 10 pg/ml AY, was added to competent SB-871 (met-6 iIu-5), which was then incubated in C-2 for four hours at 30" in the dark. At this point the cultures were plated. The table shows the colony counts per 0.2 ml and the results of analysis of the transformant and revertant colonies, by replica plating, to determine whether any cells had besn simultaneously transformed and reverted. 28 CHARLES R. STEWART

TABLE 3 Suruey of mutation reversion by acridine yellow

Revertant frequency Spontaneous after 4 hrs incubation Mutation Strain Induced by revertant frequency with 10 fig/ml.4Y

ilu-8 SB-752 Spont. * 1.3 x 10-6 3.2 x It6 dY-3 SB-753 Spont. < 10-8 < 10-8 met-6 SB-754 Spont. 2.5 x 10-6 2.3 x 10-2 met-11 SB-755 Spont. 1.3 x 10-6 1.2 x 10-5 met-12 SB-756 Spmt. < 10-8 < 10-8 arg-1 SB-757 Spont. < 10-8 < 10-8 md-1 SB-32-2t Spont. or AY* 5.1 x 10-6 4.1 x 10-6 ad-2 SB-32-3t Spont. or AY 1 x 10-8 1 x 10-8 try-2 SB-202 < 10-8 < 10-8 arg-14 SB-397 NG* < 10-8 < 10-8 thr-6 SB-399 NG 1.5 x 10-8 1.8 x 10-8 his-I? SB-400 NG I x 10-7 1 x IO-' met-13 SB-409 NG 2.6 x 10-6 3.0 x 10-6 met-7 SB-46 HNO,' < 10-8 < 10-8 leu-9 SB-271 HNO, 1.1 x 10-6 8.0 x 10-7 tyr-4 SB-272 HNO, 1.2 x 10-8 1.7 x 10-8 tyr-5 SB-398 HNO, < 10-8 < 10-8 met-14 SB-331 HNO, 2.1 x 10-6 1.4 x 10-8 his-2 SB-202 uv* 1 x 10-8 1 x 10-8 tyr-1 SB-202 uv 3.1 x 10-8 5.1 x 10-8 his-32 SB-331 UV 3.1 x 10-8 1.7 x 10-8 met-8 SB-26 uv < 10-8 < 10-8 thr-7 SB-11 uv < 10-8 < i0-8 try-11 SB-11 uv 1 x 10-8 < 10-8 arg-5 SB-230 uv 1.3 x 10-8 1.1 x 10-8 ilv-7 SB-258 uv < 10-8 < 10-& ilu-5 SB-285 uv < 10-8 < 10-8 ilu-6 SB-359 uv < 10-8 < 10-8 met-30 SB-440 uv < 10-8 < 10-8

* Abbreviations for mutagenic treatments: Spont. = spontaneous; UV = ultraviolet irradiation; AY = acridine yellow; NG = nitrosoguanidine; HNO, = nitrous acid. t Strains SB-32-2 and SB-32-3 are mutants that appeared during treatment of SB-32 (his-2-) by AY. Each mutant has an amino acid d-ficiency that has not been further characterized.

The inability of acridine yellow to revert mutants induced by UV, nitrous acid, or nitrosoguanidine is in contrast to the results of ZAMPIERIand GREENBERG (1965) and thus supports their suggestion that the high acridine concentrations that they used have effects that are qualitatively different from those of lower concentrations. The fact that maximum levels of mutagenesis can be obtained with non-competent cells and without exogenous DNA, combined with the failure to observe any association between mutagenesis and recombination of linked markers, is most easily interpreted by saying that mutagenesis in B. subtilis by acridine yellow does not require recombination. However, there are several mechanisms that MUTAGENESIS BY ACRIDINE YELLOW 29 could be postulated involving mutagenesis associated with a recombinational process that is not detected in our assay. These include: (1) It is conceivable that the process by which AY induces a mutation is the same process that inhibits transformation. Thus, the mutagenic process may be associated with one of the early steps in recombination, but its very Occurrence may prevent the completion of the recombination anld, thus, the detection of any recombinants associated with the mutants. (2) If the I-ecombination process is polarized and AY induced mutagenesis can only occur at one end of the recombining region, it would be expected that the mutations would only be correlated with recombination for markers on one side of the mutant marker. If this is the opposite side from that where the transformed (ilu) markers are located, the observed results would be expected. (3) Mechanisms are conceivable by which AY induces mutation in one strand in conjunction with recombination in the other. If the two strands are permitted to segregate before plating, the mutant and the recombinant would not be found in the same colony (the replica plating procedure detects the presence of met+ cells in an ilu+ colony, and vice versa). The failure to observe any colony that is both mutant and recombinant means that this segregation had to occur in every case. This is unlikely in view of the very slow growth that occurs in the AY culture, the fact that the frequency of revertants is increasing rapidly right up to the time of plating, and the fact that DNA synthesis in transformed cells is delayed relative to that of the rest of the population (BODMER1965; MCCARTHY and NESTER1967). (4) Recombination between the several endogenous genomes cannot be ruled out. Several workers (e.g., WEBBand KUBITSCHEK1963; MAGNI,VON BORSTEL and SORA1964) have observed that certain acridines are antimutagenic under certain conditions in Escherichia coli and in Saccharomyces cereuisiae. Since the organisms and mutations studied, the conditions of growth, and the particular acridines used were all different from those in the present study, this paper should in no way be taken as an argument against the possibility that certain acridines may have antimutagenic effects on certain mutations in B. subtilis. There is an apparent difference between the results reported here for the met-6 revertants without AY and YOSHIKAWA’S( 1966) report that the addition of transforming DNA to competent cells caused substantial increases in the reversion rate of two markers. However, the markers studied here had much higher spontaneous reversion rates than those studied by YOSHIKAWA,so that effects of the magnitude that he observed would not be noticeable here. Similarly, YOSHIKAWA’Sobserva- tion that several histidine mutants appeared in correlation with transformation of the linked try-2 marker required more sensitive techniques than those em- ployed here. It should be emphasized that the experiments presented here are by no means intended as evidence that the occurrence of a recombinational event has no influence on the probability that a mutation will occur. The contention is, rather, that acridine yellow induced mutagenesis does not require recombination.

This work was performed during the course of graduate study under the supervision of PROFESSORJOSHUA LEDEXBERG.I am grateful to DR.LEDERBERC for his support and guidance and to A. T. GANESANand L M. OKUN for many valuable discussions. 30 CHARLES R. STEWART

SUMMARY Mutagenesis by acridine yellow has been demonstrated in B. subtilis. Successful mutagenesis requires neither light nor bactericidal levels of the dye. Maximum levels of mutagenesis can be obtained with non-competent cells and without the addition of exogenous DNA. Furthermore, with mutagenesis of competent cells, no association could be observed between acridine yellow induced reversion of a particular marker and recombination for closely linked markers. These results are taken as arguments against recombination associated mechanisms of mutagenesis by acridine yellow. Reversion by acridine yellow was attempted for a number of mutants that had arisen spontaneously or had been induced by UV, nitrosoguanidine, or nitrous acid. Only with some of the spontaneous mutants was the acridine yellow able to raise the reversion rate significantly above the spontaneous reversion rate.

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