Mutation Research, 60 ( 1 9 7 9 ) 1 - - 1 1 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

PHOTODYNAMIC E F F E C T S OF DYES ON BACTERIA III. MUTAGENESIS BY ACRIDINE O R A N G E AND 500-NM MONOCHROMATIC LIGHT IN STRAINS OF ESCHERICHIA COLI THAT D I F F E R IN R E P A I R CAPABILITY *

B R U C E S. H A S S ** a n d R O B E R T B. W E B B

Division of Biological and Medical Research, Argonne National Laboratory, Argonne, IL 60439 (U.S.A.) 28 F e b r u a r y 1 9 7 8 ) ( R e v i s i o n r e c e i v e d 18 O c t o b e r 1 9 7 8 ) ( A c c e p t e d 30 O c t o b e r 1 9 7 8 ) (Received

Summary In the presence of acridine orange (AO) and monochromatic 500-nm light, the recombination-deficient strain of Escherichia coli, WP10 (recA), showed a 15-fold increase in mutation rate over the wild-type (WP2) strain. Under the same conditions, strain Bs_ 1 (uvrB lexA lon) showed a 5-fold increase in mutation rate over strain WP2. In contrast, the endonuclease-deficient strain, WP2s (uvrA), showed a lower AO-500 nm mutation rate than wild-type. The extremely high mutation rate of the recA strain cannot be due to error-prone inducible SOS repair since the inducible recA ~ function is absent. Repair of the AO-500 nm-induced lesions is likely due to a recA÷-dependent, error-free, recombination process. It is concluded that the high mutation rates with AO-500 nm light obtained in chemostat cultures of recA and lexA strains occur as a consequence of errors during semi-conservative DNA replication in the presence of unrepaired DNA lesions.

Repair of damage to the genetic material generally has been classified into 4 generic categories: [1] photoreactivation [36], [2] excision repair [10], [3] recombination repair [36], and [4] postreplication, SOS-inducible repair * Work supported by U.S. Department of Energy. ** To w h o m correspondence should be addressed. T h e s u b m i t t e d m a n u s c r i p t has b e e n a u t h o r e d by a contractor of the U.S. Government under contract No. W-31-109-ENG-38. Accordingly, The U.S. Government retains a nonexclusive, royaltyfree l i c e n s e to publish o r r e p r o d u c e t h e published form of this contribution, or allow others to do so, for U.S. Government purposes.

[25,38]. Recently, data supporting an error-free postreplication-repair pathway have been reported by Mount et al. using UV radiation on phage h [22] and Green and co-workers using UV and ethyl methanesulphonate on Escherichia coli [9]. In addition, a new pathway for DNA repair has been identified by Samson and Cairns using N-methyl-N'-nitro-nitrosoguanidine on E. coli [27]. We report here the error-free rec+-dependent repair of DNA following genetic damage by a p h o t o d y n a m i c mechanism in E. coli. The DNA lesions subject to this rec ÷ repair are produced by monochromatic 500-nm light at low fluence rates in the presence of acridine orange (AO) in cells grown in continuous (chemostat) culture. Peacocke and Skerett [23] and Beers et al. [2] have reported the formation of two types of complexes between AO and DNA and AO and polyadenylic acid (see review by Georghiou [8]). Based on these kinds of studies a general explanation has developed that acridines form two kinds of complexes with DNA. Lerman [ 17--20] has proposed that the stronger (internal) binding mode is due to intercalation of the dye molecules between adjacent base pairs of the DNA duplex; the weaker (external) binding mode, which forms at higher dye concentrations, is thought to be caused by electrostatic interactions of the dye molecules with DNA phosphate residues. Bradley and Wolf showed [3] that the 492-nm absorption band of AO shifts to about 504 nm when bound to DNA at low dye to DNA concentrations. Free AO in moderate concentrations displays a weak fluorescent emission band at 515 nm; the AO strongly bound to DNA also shows a fluorescent band at 515 nm but with a stronger peak [26]. The weak AO--DNA complex develops and absorption band at 464 nm with increasing concentration [3] and shows fluorescent emission at 620 nm [26]. The experiments described in this paper deal with a low concentration of AO and monochromatic 500 nm light in order to deal as far as possible with the intercalated dye complex. The chemostat technique was used because it is capable of measuring mutagenesis with concentrations and dosages of mutagens that produce little or no cell killing as well as yielding mutagenic data in the form of a convenient, time-dependent rate. Another report will deal with the same cells in AO irradiated with 460-nm monochromatic light. Ito [13,14] has reported differences in mutation induction with nongrowing diploid yeast in suspension when irradiated in the presence of AO with monochromatic radiation at 470 nm or 510 nm. Materials and Methods Bacterial strains The strains used are described in Table 1 : W P 2 (B/r trp) wild-type; WP2s (B/r uvrA trp), endonuclease deficient; WP10 (B/r recA-1 trp), deficient in recombination repair. In general, rec strains are very sensitive to far-UV radiation and very resistant to'mutagenesis by far-UV radiation [37]. Strain WP10 shows an F37 of 0.35 J/m 2 Strains WP2 and WP2s show F37 values of 42 and 2.0 J / m 2 respectively. (M.S. Brown, personal communication.) Some preliminary studies with several K12 AB strains are included in this report; characteristics of these strains are also indicated in Table 1.

TABLE 1 REPAIR, N U T R I T I O N O F E. coll S T R A I N S Strain

WP2 WP2s WP10 Bs_ 1 KI2 ABII57

AND

Other designations

B/r WP2Hcr B/r Hcr B / r recA

MUTATION

(SPONTANEOUS

AND

Characteristics

AO-DARK)

CHARACTERISTICS

M u t a t i o n rates a

Repair

Nutrition

Spontaneous ( m u t a n t s 1 0 -8 cells d a y - I ) b

AO-dark ( m u t a n t s 1 0 -8 cells d a y - I ) b

wild-type uvrA

trp trp

19 ( 7 . 3 ) 5 (6.0)

20 (5.2) 59 ( 5 . 6 )

recAl l e x A l O 1 uvrB wild-type

trp

13 (8.5) 38 (8.8) 45 (5.0)

37 ( 5 . 2 ) 58 (6.0) 45 (5.0)

17 ( 3 . 5 )

44 (6.0)

24 (4.9)

91 (7.3)

35 (6.6)

55 (5.0)

K12 AB1886

uvrA6

K12 AB2463

recA13

K12 AB2480

recA13 uvrA6

his a r g p r o thr leu thi his a r g p r o thr leu thi his arg pro thr leu thi pro thi

a Values e x c e p t for strains B s - I and K 1 2 A B 2 4 8 0 a r e t a k e n f r o m R e f . I I . b N u m b e r s in parenthesis are m e a n n u m b e r o f generations per d a y .

Culture conditions The E. coli cultures were grown in glucose-limited chemostates [15,16]. The

glucose concentration was relatively low (50 mg/1) to reduce the cell concentration under the assumption that the number of spontaneous and induced mutations in a faster growing state under chemostat conditions is a function of cell concentration. The growth medium also contained 0.5 mg/1 concentration of tryptophan in M-9 salt solution [1]; the temperature was maintained at 37°C. Acridine orange (Aldrich} was used at 2 × 10 -6 M concentration. The growth conditions of these experiments were exactly the same as those reported earlier by Hass and Webb [11]. Mu tan t assay

As in the AO-dark experiments [11], resistance to phage T5 (T5 R) was used as the mutation endpoint. The assay for T5 R mutants was performed b y spreading quadruplicate samples of the treated bacteria in the presence of excess T5 phage on iron-supplemented nutreient agar (Difco) plates [16]; total counts were obtained from the same samples after appropriate dilution and counted after 24--48 h incubation at 37°C. The mutation rate was derived from a leastsquares fit to plots o f mutation frequency versus time. Except for prescribed irradiation, all chemostat cultures were grown in the dark with subsequent handling being done under General Electric F 4 0 / G O laboratory safe lights operating at very low fluence rates. Irradia tion

The chemostat apparatus was that described by Kubitschek [15] except that the growth vessel was rectangular in cross-section with an inside light path of

i

o "500" B-I FILTER • GROWTH MEDIUM: AO(2xlO-6M),I GLUCOSE (50mg/I), TRYPTOPHAN I (0.5rag/I), M-9 SALTS J

0.12

--'100 9O

0.10

8o ..~

A

a 0.06

50

,_,'~ ~0.o4

4o 3o~

0.02

I--

20 v_



I0 0

22000 20000 WAVENUMBER (cm -I ) I 440

I

I

I

I

18000 I

0

I

460 '$80 500 520 540 560 WAVELENGTH (nm)

Fig. 1. A b s o r p t i o n o f the c h e m o s t a t g r o w t h m e d i u m c o n t a i n i n g 2 X 10 -6 M A O ( e ) and t r a n s m i s s i o n o f the filter p r o d u c i n g m o n o c h r o m a t i c 5 0 0 - n m light (©). T h e a b s o r p t i o n bands w e r e p a r t i t i o n e d by a s s u m i n g that the " 5 0 0 " band is s y m m e t r i c a l ( o n the e n e r g y scale) a b o u t its p e a k at 4 9 7 n m . T h e 4 6 0 - n m band is t h e n c o n s t r u c t e d by s u b t r a c t i o n f r o m the e x p e r i m e n t a l curve. T h e h a t c h i n g r e p r e s e n t s the f r a c t i o n o f light t h a t t h e 5 0 0 - n m filter a l l o w s to pass that is c o m m o n to the 4 6 0 - and 5 0 0 o h m bands.

1 cm. Visible radiation was provided by a standard 35-mm side projector, equipped with a 400-W Sylvania (CYK 200 h) bulb operating from a constantvoltage transformer. During the course of a single experiment (3 days) radiant emission declined no more than 10%. The beam was filtered with a Baird-Atomic B-1 interference filter blocked on the low wavelength side to X-rays and on the high side to "infinity ;" wavelength of maximum transmission was 500 nm with a band width at 10% transmission of 9 nm (Fig. 1). In the preliminary experiments, the beam was filtered with a Baird-Atomic B-1 interference filter with a transmission peak at 490 nm and a band width a 10% transmission of 9 nm. Dosimetry was performed before each experiment using a Hilger--Watts Model 17.1 thermopile (previously standardized against a National Bureau of Standards lamp) and a Keithley microvoltometer. Fig. 1 shows the absorption spectrum of AO decomposed into the "460" and "500" nm component absorption bands; the transmission characteristics of the 500-nm B-1 filter are also shown. The area of intersection of the 500-nm B-1 filter and the 460--AO absorption band is 5%. With the 490-nm B-1 filter, the intersection with the 460--AO absorption band is less than 10%. Results The results of AO-light studies with E. coli K12 strains which represent our initial experiments with AO and monochromatic light are summarized in

1500

I

I

I

I

2FM AO 1250 _A

u~ I000 ¢g,

-

o n

I

/

WPIO: 0.29 Wm -2 BS_I: 0.84 Wm-2

#f /

• W P 2 : 0 . 8 4 Wm -2 a WP2s: 0.84 Wm -2 =1 = Mean of two determinations

-

/

/~recAI /

/

- -

G0

o LLI [1. O3 I'-Z

N

750 -

50Ohm LIGHT

5oo

AxA

// /

I

uvrB -

/

/

/

-

-

Wild T y p e - , . ~ /

P'





• I

l

25O ~

,

e

~

"

-

W

i

l

0 -I

. . . . . . . . .

d

0

Type, Dark (5 gen./doy)

I

I

I

I

2

3

TIME, days

call

Fig. 2. Typical chemostat mutant-production responses in various E. strains possessing different repair capabilities irradiated continuously by 500-nm light in the presence of 2/zM AO. Cells were constantly exposed to AO; irradiation at 500 nm was begun at day zero on the time axis. See text f o r details.

Table 2. The K12 recA strain (AB2463) showed a mutation rate to phage T5 resistance 11 times that of the wild-type K12 strain ( A B l 1 5 7 ) . The A O - - 4 9 0 nm mutation rates of K12 AB2463 (recA) and K12 A B 2 4 8 0 (recA uvrA) axe not significantly different, indicating that the absence of the excision-repair endonuclease (uvrA) does not increase the mutation rate in the K12 recA strains. These results prompted us to do experiments with the WP strains in TABLE 2 S U M M A R Y O F R E S U L T S O F C H E M O S T A T M U T A T I O N ( T 5 R) O F E. coli K 1 2 S T R A I N S IN T H E P R E S E N C E O F A O (2 X 1 0 -6 M) A N D M O N O C H R O M A T I C 4 9 0 - n m L I G H T a Strain

AO---dazk mutation rate b ( m u t a n t s 10 -8 cell d a y -1 )

AO--490 nm mutation rate observed ( m u t a n t s 10 -8 cell d a y -1 )

Averaged normalized AO--490 nm mutation rate ( m u t a n t s 10 -8 ceil d a y -1

R a t i o of A O - - 4 9 0 nm mutation rate t o t h a t of w i l d - t y p e (ABl157)

W-! m-2)

AO--490 nm mutation rate corrected for spontaneous and A O---dark r a t e s ( m u t a n t s 10 -8 d a y - 1 W-1 m - 2 )

K 1 2 AB1157 KI2 AB2463

40 20

357 3600

238 2258

198 2238

1 11

K12 AB2480

55

4089

2580

2525

13

a L i g h t f r o m 4 0 0 - W S y l v a n i a C Y K 2 0 0 h b u l b f i l t e r e d w i t h B a l r d - A t o m i c B-1 i n t e r f e r e n c e filter w i t h m a x i m u m t r a n s m i s s i o n a t 4 9 0 n m a n d a b a n d w i d t h a t 1 0 % t r a n s m i s s i o n o f 9 n m . T h e f l u e n c e r a t e w a s 1.6 W m -2 . G l u c o s e a n d r e q u i r e d a m i n o acid ( T a b l e 1) c o n c e n t r a t i o n s w e r e 1 0 0 rag/1 a n d r e s p e c t i v e l y 5 mg/l. b T h e AO---dark r a t e s i n c l u d e t h e s p o n t a n e o u s m u t a t i o n rates. G r o w t h rates were 4--6 generations per day.

1.75 0.85

0.84 1.04

0.29

uvrA

wild-type

recA-1

l e x A uvrB

WP2s

WP2

WP10

Bs__1 515 605

1000

210 168

330 137

Observed mutation rate ( m u t a n t s 10 -8 ceB d a y - l )

MUTATION

1061

3024

220

180

Normalized b averaged mutation rate ( m u t a n t s 10 -8 cell d a y - l W -1 m - 2 )

OF CHEMOSTAT

58

37

20

59

Mutation rate of A O - e x p o s e d cells in t h e d a r k c ( m u t a n t s 10 -8 cell d a y -1 )

OF AO

1003

2987

200

121

Mutation rate corrected for spontaneous and A O--dark rates c ( m u t a n t s 10 -8 cell d a y -1 W -1 m - 2 )

( T 5 R ) O F E. coli S T R A I N S I N T H E P R E S E N C E

a C o r r e c t e d f o r e n e r g y a b s o r p t i o n b y A O . T h e O.D. o f A O a t 2 X 10 -6 M is 0 . 1 5 8 . H e n c e t h e f l u e n c e is c o r r e c t e d b y m u l t i p l y i n g b y 0 . 8 4 ( J . J a g g e r , I n t r o d u c t i o n t o R e s e a r c h in U l t r a v i o l e t P h o t o b i o l o g y . P r e n t i c e - H a l l , N e w J e r s e y , 1 9 6 7 , p . 1 3 2 . b Normalized by dividing observed m u t a t i o n rate b y the fluence rate. c See R e f . 11 e x c e p t f o r s t r a i n B s _ l . I t is a s s u m e d t h a t t h e s p o n t a n e o u s m u t a t i o n r a t e is a c o m p o n e n t o f t h e A O - - d a r k m u t a t i o n r a t e .

0.84 0.50

Corrected a flnence rate ( W m -2 )

Repai~ characteristics

E. coli strain

SUMMARY OF EXPERIMENTAL PARAMETERS AND RESULTS (2 X 1 0 - 6 M) A N D M O N O C H R O M A T I C 5 0 ( N n m L I G H T

TABLE 3

which we have characterized the AO--dark mutation rates [11]. In order to minimize the overlap of the two AO-absorption bands (see Fig. 1) we irradiated the WP strains at 500 nm instead of 490 nm. The results of typical chemostat experiments with E. coli WP (B/r) strains are shown in Fig. 2. The complete data set is described in Table 3. In terms of the normalized mutation rate (uncorrected for AO--dark mutagenesis) the wildt y p e strain WP2 was slightly, b u t significantly, more susceptible to AO--500 nm mutagenesis than was the uvrA endonuclease-deficient (WP2s) strain. The correction for AO--dark mutagenesis increased the factor by 30%. The high mutation rate obtained with recA strain (24.7 times the mutation rate of the uvrA strain} was unexpected. These data are in marked contrast with the mutation rates produced in cells of the same recA strain by far-UV radiation and by a number of chemical mutagens. While uvrA strains show high mutability by far-UV radiation [4,39], recombinationless strains (e.g., WP10) show very low susceptibility to UV-induced mutations [21,37], although they are quite sensitive to inactivation by far-UV radiation. Whatever other repair systems possessed by these recombination-less strains, the repair systems are error-free in their mode of action when far-UV is the lethal or mutagenic agent. A mutation rate to T 5 R much greater than in wild-type (WP2) or the endonuclease-deficient (WP2s) strains was obtained also with chemostat cultures of E. coli Bs_ 1 (lexA uvrB lon) (Fig. 2, Table 3). Both strains Bs-1 and WP10 are refractory to mutagenesis by far-UV radiation. We previously described mutagenesis in chemostat cultures of these strains in the presence and absence of AO in the absence of light [11]. A comparative summary of these results is shown in Table 4, and comparisons of A O - 5 0 0 nm mutation rates with AO--dark rates and with spontaneous rates also are listed. While the uvrA strain (WP2s) showed the largest ratio of mutation rates in the AO--dark to spontaneous comparisons (12X), it was the recA strain that showed the largest increments in the AO--500 nm to AO--dark (81X) and AO- 500 nm to spontaneous (230X} categories. This relationship implies a different mechanism of action and different lesions when light is present than when it is absent. Light in the absence of AO was not mutagenic at wavelengths of TABLE 4 RELATIONSHIP BETWEEN SPONTANEOUS, AO--DARK, AND AO--500 nm MUTATION RATES OF S T R A I N S O F E. coli B A N D B]r W I T H D I F F E R I N G R E P A I R C H A R A C T E R I S T I C S

WP2s WP2 WP10 Bs--1

AO--dark minus spontaneous a mutation rate (mutants 10 -8 cell d a y - 1 )

Ratio of A O - - d a r k to spontaneous mutation rate

Ratio of AO--500 nm b to AO--dark mutation rate c

R a t i o of AO--500 nm b spontaneous mutation rate c

Ratio of AO--500 nm mutation rate with that of WP2 b

54 1 24 20

12 1 3 1.5

2 10 81 17

24 11 230 26

0.6 1 15 5

a S e e T a b l e 1 a n d s e e R e f . 11. b A O - - - 5 0 0 n m m u t a t i o n rate a f t e r s u b t r a c t i o n o f A O - - d a r k m u t a t i o n r a t e . c Ratio compares irradiation-normalized mutation rate (mutants 10 -8 cells day -I W -I m -2) to spontaneo u s o r A O - - d a r k mutation rate (mutants 10 -8 cells day-l). All values are normalized to 2 p M A O .

500 nm and above in E. coli B/r ( t o n B trp) (35) or E. coli B/r (trp ÷) (unpublished data). Discussion

The decrease in mutagenesis by AO plus 500-nm light in the u v r A strain (Fig. 2, Table 3), is similar to that found in a Salmonella strain by Imray and MacPhee [12] using methylene blue and broad-spectrum visible light at doses that produced considerable cell killing. However, in contrast to the data we report here, the same study [12] reported a decrease in mutagenesis in a rec strain. The fact that the wild-type strain (WP2) was mutated significantly more by AO--500 nm action than was the u v r A strain (WP2s) indicates that the type of lesion produced does n o t require the u v r A endonuclease for repair. Indeed, the presence of this enzyme may cause competition or interference with enzymes that do perform efficient (error-free} repair of AO--500 nm produced DNA lesions. R e c A strains lack the rec ÷ function that is essential for both the error-free recombination repair and the inducible error-prone " S O S " repair systems [25,38]. Thus AO--500 nm mutagenesis presumably occurs either in a r e c A ÷ dependent error-prone prereplication-repair process or from errors during semiconservative DNA replication as a result of unrepaired (prereplication) DNA lesions. It is unlikely that the mutational lesions produced by AO--500 nm light include pyrimidine adducts since the known pyrimidine adducts (including cyclobutane dimers) do not require oxygen for production. Furthermore, no p h o t o p r o d u c t s of this kind have been reported for AO--light interaction. Therefore, other lesion candidates must be sought to account for AO--500 nm mutagenesis. Since methylene blue in the presence of broad-spectrum visible light (VL) efficiently and selectively disrupts the structural integrity of guanine in DNA [31], a similar t y p e of damage to nucleoside bases by AO and light was hypothesized. Although an early report found AO--VL to have no destructive effect on guanine [32], later experiments did show photolytic disruption of guanosine [29] and viral RNA [28] by AO--VL, b u t at doses and concentrations that were much greater than those required for methylene blue effects. Georghiou [7] has made a case for preferential electronic interactions of proflavine (PF, and AO analog) with guanine residues. However, since Schreiber and Daune [30] in examining PF- and AO-complexes with polydeoxyribonucleotides emphasize that the interaction of PF is not identical with that of AO, caution must be employed when comparisons are made. Freifelder and Uretz [6] found that AO--VL produced an alkali-labile bond that was probably some form of base damage associated with the N-glycosidic bond. However, continuation of the work of Freifelder et al. [5] by Petrusek indicated that both true single-strand breaks and alkali-labile bonds are produced in DNA by AO--VL [24]. Another p h o t o d y n a m i c DNA lesion has been reported by Smith [33], who found that AO--VL decreases the extractability of DNA in irradiated cells presumably due to DNA--protein crosslinks. Petrusek [ 24] concluded that such a DNA--protein crosslink may play an important role in AO--VL inactivation;

b u t aside from the apparent absence of repair of this lesion, the extent of the role of DNA--protein crosslinks, particularly as a p h o t o d y n a m i c mutagenic lesion, remains undetermined. Regardless of the t y p e of DNA lesions reported, all of the experiments cited above were performed with broad-spectrum VL sources, including wavelengths in the 450--550 nm range. The results of those experiments are therefore ambiguous, since in striking contrast to the AO--500 nm results. AO--460 nm light was less mutagenic in the r e c A strain than in the wild-type (unpublished data). Therefore, different wavelengths produce dramatically different effects and, therefore, probably different lesions. Also, these data support the earlier conclusion [34] that dye intercalation in DNA plays a major role in photodynamic mutagenesis for cells growing in continuous culture. Whatever the chemical nature of the AO--500 nm lesions, their mutagenic action is strongly reduced by a repair process that requires the presence of the rec ~ gene p r o d u c t (Table 3). The r e c ÷ - d e p e n d e n t repair must be relatively errorfree because the u v r A r e c ~ strain (WP2s) shows a 25-fold lower mutation rate than the r e c A strain (WP10). At the same time, the high rate of mutation in the r e c A strains cannot occur as errors during typical r e c k - d e p e n d e n t SOS repair because this type of repair cannot occur in r e c A strains. Since the WP2s ( u v r A rec ~) strain is m u t a t e d at a lower rate than wild-type by AO--500 nm light (Table 3), we conclude that the error-free repair of AO--500 nm (and AO--490 nm) lesions does not require the u v r A endonuclease. Lethal lesions produced b y AO--500 nm light at much greater fluence rates than those used in the mutation studies are strongly repaired in rec ÷ l e x ÷ strains (wild-type and u v r A ) g r o w n in chemostat cultures; r e c A and l e x A strains were 50--175 times more sensitive to lethal action than wild-type and u v r A strains. In parallel with the mutation results, u v r A strains are not more sensitive than wild-type strains to 500-nm light (unpublished data). We propose that AO--500 nm (and AO--490 nm) mutagenesis occurs as a consequence of the presence of unrepaired DNA lesions during normal semiconservative DNA replication. Further, these mutational lesions can to a large extent be repaired efficiently by a relatively error-free process that requires a functional r e c ÷ l e x ÷ repair system, b u t does n o t need the u v r A endonuclease. Nonetheless, if postreplication gap filling is involved in the repair of AO--500 nm (or AO--490 nm) DNA lesions, it likely occurs by the recombinational (error-free) rather than the inducible (error-prone) SOS process. The existence of such an error-free process has been suggested by Witkin [38], and evidence for it in a phage X-E. coli tsl r e c A system was presented b y Mount and co-workers [22] and b y Green and co-workers [9] with E. coll. Acknowledgement The authors wish to acknowledge with thanks the gift of strain WP10 from Dr. Evelyn M. Witkin. The authors acknowledge the technical efforts of Miss Mary Oskowski.

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Photodynamic effects of dyes on bacteria. III. Mutagenesis by acridine orange and 500-nm monochromatic light in strains of Escherichia coli that differ in repair capability.

Mutation Research, 60 ( 1 9 7 9 ) 1 - - 1 1 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press PHOTODYNAMIC E F F E C T S OF DYE...
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