624
Biochimica et Biophysica Acta, 544 (1978) 624--633 © Elsevier/North-Holland Biomedical Press
BBA 28726
PHOTODYNAMIC INACTIVATION AND ITS REPAIR IN MYCOPLASMAS
UTPAL CHAUDHURI,JYOTIRMOY DAS and JACK MANILOFF * Department of Microbiology, University of Rochester, Medical Center, Rochester, N.Y. 14642 (U.S.A.) (Received May 8th, 1978)
Summary Photodynamic inactivation is the loss in viability observed when organic dyetreated cells are exposed to visible light and molecular oxygen. The photodynamic inactivation of mycoplasmas, the smallest free living cells, has been studied. Depending on the extent of inactivation in Acholeplasma laidlawii, photodynamic induced damage can be repaired if the irradiated cells are incubated in the dark in buffer. Analysis of the DNA of these cells shows that photodynamic inactivation induces single strand breaks which can be repaired during liquid holding. To examine possible damage to the cell membrane, glucose uptake was studied as a permeability measure. Neither acriflavine nor photodynamic inactivation had any measurable effect on membrane permeability.
Introduction Aminoacridine dyes have antibacterial and antiviral effects and can induce frameshift mutations. When acridine treated bacterial cells are exposed to visible light and molecular oxygen there is a loss of viability, referred to as photodynamic inactivation [1,2]. This inactivation is believed to be primarily due to acridine dye-DNA interactions [2], although both proteins and cell membranes have been suggested to also be involved in photodynamic inactivation [3--6]. DNA lesions are known to occur during photodynamic inactivation and three possible models for this effect have been proposed: (i) base modification [7]; (ii) single-strand breaks [8,9]; and (iii) cross-linking of DNA and pro~ins [10]. Most studies have not explicitly examined the possible repair of photodynamically induced DNA damage, which has led to some controversy as to whether this damage can be repaired [ 1 1 - 1 3 ] . Das et al. [14] investigated the * T o w h o m r e p l l n t r e q u e s t s s h o u l d be a d d r e s s e d .
625 liquid-holding repair capabilities of radiation resistant Escherichia coli B/r and radiation sensitive E. coli BB-1 strains after photodynamic damage and showed that, while the B/r strain can repair such damage, the Bs-1 strain lacks this capability. The polA and rec genes have been found to have a role in repair of photodynamic damage [9,15,16]. In recent studies the repair capabilities of the mycoplasmas have been examined. These prokaryotes are the smallest free living cells. They do not have cell walls and each cell is bounded by a single lipoprotein membrane [17]. Acholeplasma laidlawii, a mycoplasma with a genome size of 1 • 109 daltons, has been shown to have both dark (excision) and light repair mechanisms for the repair of ultraviolet light-induced DNA damage [18]. Mycoplasma gaUisepticum, a mycoplasma with a genome size of 0.5 • 109 daltons, was found to lack both of these repair mechanisms [19]. Treatment of ultraviolet light-irradiated A. laidlawii with the acridine dye acriflavine inhibits excision repair, but acriflavine treatment before irradiation partially protects the cells against inactivation [20]. Acriflavine treated A. laidlawii cells also show reduced host cell reactivation of ultraviolet light-irradiated mycoplasmaviruses [21]. We report here on studies of photodynamic inactivation and its repair in mycoplasmas. These studies show that A. laidlawii can repair some photodynamic inactivation damage to its DNA and that cell permeability is not affected. Materials and Methods
Cells, media and buffers. The mycoplasmas used in this study were Acholeplasma laidlawii strain JA1 and Mycoplasma gallisepticum strain A5969. These are the strains used in previous DNA repair studies reported from this laboratory [19--21]. A. laidlawii cells were grown in tryptose broth and assayed on tryptose agar plates as previously described [22]. M. gallisepticum cells were grown in mycoplasma broth base (MBB) medium and assayed on MBB agar plates as described previously [19]. ~-Buffer was 0.05 M trishydroxymethylaminomethane (Tris), 0.156 M NaC1 and 0.01 M ~-mercaptoethanol, pH 7.4. The holding buffer was one part growth medium and nine parts ~-buffer. Acriflavine treatment. Acrifiavine hydrochloride was obtained from Sigma Chemical Corp. (St. Louis, Mo.). Acriflavine solutions (1 mg/ml) were made in sterile distilled water and stored in the dark at 0--4°C. The acriflavine solution had an adsorption maximum at 445 nm and molar extinction coefficient of 3.4-104 M-1 at this wavelength. For acriflavine treatment, exponentially growing cells were harvested by centrifugation (8000 rev./min for 10 min) and resuspended (to a final titer of 1--2 • l 0 s colony-forming units/ml) in holding buffer, containing acriflavine at a final concentration of 0.5 pg/ml. The cell suspension was incubated in the dark for 1 h at 37°C. Irradiation o f cells. 2-ml cell suspensions, either untreated or treated for 1 h with acriflavine were put in 60-mm petri dishes and irradiated with a 40-W fluorescent lamp at a distance of 14 cm. The cell suspension was kept in an ice bath during irradiation and stirred ocasionally. At different times, 0.1-ml samples were removed and assayed for colony-forming units. All operations after
626 irradiation were carried out in dim light. Acriflavine solutions irradiated this way for 60 min showed no detectable change in absorbance at 445 nm, indicating that any bleaching of the dye was insignificant during these experiments. Liquid holding recovery. Samples of irradiated cells were diluted 100-fold in holding buffer and incubated in the dark at 37°C. At different times of incubation samples were removed and assayed for colony-forming units. Macromolecular synthesis. To measure DNA, RNA or protein synthesis, [3H]deoxythymidine (20 ~Ci/ml), [3H]uridine (20 pCi/ml) or [14C]leucine (10 gCi/ml) was added, respectively, to a cell suspension. At different times duplicate samples were removed and assayed for trichloroacetic acid precipitable radioactivity as described previously [23]. Glucose uptake. Glucose transport in acriflavine treated cells was examined following the method of Rottem and Razin [24]. For these experiments the buffer was prepared as follows: to nine parts of a buffer containing 0.05 M Tris-HC1, 0.15 M NaC1 and 0.01 M MgC12 (pH 8.0) was added one part of tryptose broth containing neither glucose nor pleuropneumonia-like organism (PPLO) serum fraction [3.0.methyl-3H]-D-Glucose (25 pCi(ml) (New England Nuclear, Boston, Mass.) was added to cell suspensions in this buffer and thecells were incubated in the dark at 37 ° C. At different times after addition of the labelled glucose, 50-pl samples were withdrawn and filtered. The filters containing cells were washed, dried and assayed for radioactivity. Sedimentation in alkaline sucrose gradients. Control and photodynamically inactivated cells were analyzed in 5 ml 5--20% (w/v) sucrose gradients (adjusted to pH 12.0 with NaOH) as described previously [18]. The sucrose was in ~-buffer containing 0.005 M EDTA. Sedimentation was in a Beckman SW50.1 rotor for 2 h at 28 000 rev./min. Calculation o f molecular weights. From the gradient analysis, number average molecular weights of cell DNA's were calculated relative to T4 marker DNA (included in all gradients), as previously described [ 18]. Survival curves. From the photodynamic inactivation data survival curves were drawn using a least-square data analysis by a computer program described previously [ 18]. Results
Effect o f acriflavine on cell growth and macromoleeular synthesis. Exponentially growing mycoplasma cells were transferred to prewarmed fresh medium containing different concentrations of acriflavine and incubated at 37°C in the dark for 3 h (a little over one doubling time). Cell growth (Table I) was not significantly affected by up to 1 pg acriflavine/ml. At increasing acrifiavine concentrations cell growth was inhibited and finally a decrease in cell viability was observed. M. gallisepticum was more sensitive to acriflavine than A. laidlawii. The synthesis of DNA, RNA and protein was examined in A. laidlawii cells treated with different concentrations of acriflavine. For these experiments, acriflavine and labelled precursors were added at zero time and macromolecular synthesis was measured as a function of time. DNA and protein synthesis were reduced by approx. 20%, relative to untreated control cells (Figs. l a and c), by
TABLE I EFFECT OF ACRIFLAVINE
ON MYCOPLASMA GROWTH
T h e i n i t i a l cell t i t e r o f A . laidlawii w a s 1 . 3 • 1 0 8 c o l o n y - f o r m i n g u n t t s / m l a n d o f M. g a l | i s e p t i c u m w a s 7.7 • 1 0 ~ c o l o n y - f o r m i n g u n i t s / m l . T h e f i n a l cell t i t e r w a s m e a s u r e d a f t e r 3 h i n c u b a t i o n a t 3 7 ° C in t h e da~k in v a r i o u s a c r i f l a v i n e c o n c e n t r a t i o n s . Acrlflavine (#g/ml)
Final titer/Initiai titer M. g a l l i s e p t i c u m
A. laidZawil 0 0.5 1.0 2.0 5.0 10.0
2.2 1.9 1.9 1.1 1.1 0.5
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Fig. 1. E f f e c t o f v a r i o u s a c r i f l a v i n e c o n c e n t r a t i o n s o n A. laidlawii m a c r o m o l e c u l a r s y n t h e s i s . A t z e r o t i m e , cells i n t r y p t o s e b r o t h w e r e m i x e d w i t h a e r i f l a v i n e a n d (a) [ 3 H ] d e o x y t h y m i d i n e , ( b ) [ 3 H ] u r i d i n e , o r (e) [ 1 4 C ] l e u e i n e , a n d t r i e h l o r o a e e t i c a e i d - p r e e i p i t a b l e r a d i o a c t i v i t y w a s m e a s u r e d as a f u n c t i o n o f i n c u b a tion thne at 37°C in the dark. The final acrlflavine concentrations were: o, no dye; A 0.5 #g/ml; a, 1 #g/ m l ; o, 2 / ~ g / m l ; a , 5 / z g / m l ; a 1 0 / ~ g / m l . F i g . 2. D N A s y n t h e s i s in A . loidIawli a f t e r r e m o v a l o f a c r i f l a v i n e . Ceils w e r e i n c u b a t e d i n t r y p t o e e b r o t h , c o n t a i n i n g (o) n o d y e , (z~) 0 . 5 , (m) 5 o r (J,) 1 0 # g a c r i f l a v i n e l m l , f o r 1 h a t 3 7 ° C in t h e d a r k . C a l l l w e r e centrifuged, washed once with trYPtose broth and resuspended in fresh tryptose broth containing 20 #Ci [ S H ] t h y m i d i n e / m l . T h e t i m e o f l a b e l a d d i t i o n w a s t a k e n as t h e z e r o t i m e p o i n t a n d i n c o r p o r a t i o n i n t o a c i d - i n s o l u b l e m a t e r i a l w a s m e a s u r e d as a f u n c t i o n o f tirae.
628
up to 1 pg acriflavine/ml. At higher dye concentrations, both DNA and protein synthesis were almost completely inhibited. In contrast, RNA synthesis was significantly affected even at low acriflavine concentrations (Fig. lb). This is consistent with observations that in the absence of light acridine dyes inhibit bacterial DNA dependent RNA polymerase [25,26]. Reversibility o f acriflavine inhibition o f DNA synthesis. A laidlawii cells were incubated in the dark for 1 h in tryptose broth containing acriflavine, then washed free of dye and resuspended in fresh medium containing [3H]deoxythymidine. The reduction in DNA synthesis observed in cells treated with a low (0.5 pg/ml) acriflavine concentration (Fig. la) was almost completely reversed when the dye was removed (Fig. 2). DNA synthesis was inhibited by 5 pg acriflavine/ml (Fig. la), but some resumption in synthesis was seen after acriflavine removal (Fig. 2). However, inhibition of DNA synthesis by 10 ug acriflavine/ml (Fig. la) was irreversible (Fig. 2). Since incubation in the dark in 0.5 pg acriflavine/ml gave only minimal effects on cell growth and macromolecular synthesis, this dye concentration was used in all subsequent photodynamic inactivation studies. Photodynamic inactivation o f mycoplasmas. These studies used both A. laidlawii and M. gallisepticum. Control experiments with untreated cells showed that the cells were not very sensitive to visible light: there was approx. 85% survival of colony-forming units for both A. laidlawii and M. gallisepticum after 40 min of irradiation (data not shown). The photodynamic inactivation curve of acriflavine-treated A. laidlawii (Fig. 3) after visible light irradiation had a shoulder after which inactivation was exponential. In terms of survival fractions, the zero dose intercept of the linear part of the A. laidlawii survival curve was 12. In contrast to the A. laidlawii curve, the M. gallisepticum survival curve was essentially exponential, with a zero dose intercept of 2 (Fig. 3). The presence of a shoulder in the photodynamic inactivation curve of A. laidlawii (Fig. 3) suggested the possible existence of a repair mechanism for photodynamic damage in these cells. Therefore, experiments were carried out to examine liquid holding recovery of such damage. Liquid holding recovery. To examine the kinetics of dark repair, A. laidlawii cells in holding buffer containing 0.5 gg acriflavine/ml were irradiated with visible light, diluted 1 : 100 with holding buffer and incubated at 37°C in the dark. Samples were removed and plated as a function of time. Untreated irradiated control cells incubated in holding buffer showed no change in titer for the holding times used in these experiments. Cells which were irradiated for short times (cell survival greater than approx. 40%) showed almost complete recovery if they were plated by 20 min of holding time (Fig. 4a). However, longer holding resulted in a decrease in recovery. Cells which were irradiated for longer times (cell survival less than 10%) showed no significant a m o u n t of liquid holding recovery (Fig. 4b) and a loss of cell titer with longer holding times. To measure the dependence of the exten~t of recovery on the initial a m o u n t of photodynamic inactivation, samples of cells were irradiated to different survival fractions, held (as above) for 20 min, and then plated. These data (Fig. 5) show that the a m o u n t of recovery is highly dependent on the initial a m o u n t of photodynamic inactivation.
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Fig. 3. P h o t o d y n a m i c i n a c t i v a t i o n o f a c r i f l a v i n e t r e a t e d A. lafdlawii (o) and M. gaUisepticum (~). Cells w e r e i n c u b a t e d i n h o l d i n g b u f f e r c o n t a i n i n g 0 . 5 ~ g a c r t f l a v t n e / m l a t 3 7 ° C f o r 1 h i n t h e d a r k . T h e cell s u s p e n s i o n w a s t h e n i r r a d i a t e d w i t h visible l i g h t a n d a s s a y e d f o r c o l o n y - f o r m i n g u n i t s as a f u n c t i o n o f irradiation time. Fig. 4 . R e p a i r k i n e t i c s o f A. laidlawii a f t e r p h o t o d y n a m i c i n a c t i v a t i o n . Cells i n h o l d i n g b u f f e t c o n t a i n l n g 0 . 5 # g a c r i f l a v t n e / m l w e r e i n a c t i v a t e d as d e s c r i b e d f o r F i g . 3 t o e i t h e r (a) 4 6 % o r Co) 4 % survival. T h e cells w e r e d i l u t e d I : I 0 0 w i t h h o l d i n g b u f f e r a n d i n c u b a t e d i n t h e d a r k a t 3 7 ° C . S a m p l e s w e r e r e m o v e d a n d a s s a y e d f o r c o l o n y - f o r m i n g u n i t s as a f u n c t i o n o f t i m e .
Macromolecular synthesis in photodynamically inactivated cells. To examine the effect of p h o t o d y n a m i c inactivation on macromolecular synthesis, A. laidlawii cells in holding buffer were treated with 0.5 ~zg acriflavine/ml for 1 h at 37°C in the dark and irradiated to various survival fractions. The cells were centrifuged, washed with tryptose broth and (at zero time) resuspended in prewarmed tryptose broth containing [3H]deoxythymidine, [3H]uridine, or [14C]leucine. Samples were removed and assayed for acid insoluble material as a function of time. In these experiments control cells were treated with acriflavine b u t n o t irradiated; hence these cells had 100% survival after the acriflavine treatment.
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Fig. 5. L i q u i d h o l d i n g r e c o v e r y o f A . ~ i d ~ w i i as a f u n c t i o n o f p h o t o d y n a m i c i n a c t i v a t i o n . Cells i n h o l d i n g b u f f e r w e r e i n a c t i v a t e d t o v a r i o u s s u r v i v a l f r a c t i o n s as d e s c r i b e d f o r Fig. 3. T h e cells w e r e t h e n d i l u t e d 1 : 100 with holding buffer, incubated for 20 rain in the dark at 37°C, and assayed for colony-forming units. Fig. 6. D N A , I%NA a n d p r o t e i n s y n t h e s i s i n p h o t o d y n a m i e a l l y i n a c t i v a t e d A . laidlawii. Cells i n h o l d i n g b u f f e r c o n t a i n i n g 0 . 5 # g a c r i f l a v i n e / m l w e r e i n a c t i v a t e d t o v a r i o u s survival f r a c t i o n s as d e s c r i b e d f o r Fig. 3: ( e ) I 0 0 , (A) 8 0 , (~) 5 0 , a n d ( o ) 7% survival. T h e cells w e r e c e n t r i f u g e d , w a s h e d w i t h t r y p t o s e b r o t h , a n d ( a t z e r o t i m e ) r e s u s p e n d e d i n t r y p t o s e b r o t h c o n t a i n i n g (a) [3 H ] d e o x y t h y m i d i n e , (b) [3 H ] u r i d i n e , o r (c) [ 1 4 C ] l e u c i n e . A c i d p r e c i p i t a b l e r a d i o a c t i v i t y w a s m e a s u r e d as a f u n c t i o n o f t i m e .
Under these conditions (Fig. 6), cells photodynamically inactivated to 70-90% survival immediately resumed DNA synthesis; however, there was a slight lag in the resumption of RNA and protein synthesis. In contrast to this, cells inactivated to 40--60% survival had a pronounced lag of approx. 2 h before DNA, RNA and protein synthesis resumed. If cells were inactivated to a greater e x t e n t (i.e., approx. 10 percent survival) no resumption of DNA, RNA or protein synthesis was observed, even after an overnight incubation in growth medium containing labeled precursors (data not shown). These biochemical results are in agreement with the viability recovery data (Fig. 4).
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Fig. 7. Glucose u p t a k e in p h o t o d y n a m l e a i l y inactivated A . |aidlawii. Cells i n h o l d i n g buffer e o n t - t n t n g 0.5 #g acriflavine/ml were either unirradiated (e) or irradiated w i t h visible light to 10 pe rc e nt survival (o). Cells were centrifuged, washed, and (at zero time) resuspended i n buffer c o n t a i n i n g ~ - [ 3 H ] m e t h y l glucose, and u p t a k e of radioactivity essayed as a f u n c t i o n of time, as described i n Materials and Methods. Fig. 8. Alkaline sucrose gradient analysis of [ 3 H ] d e o x y t h y m i d i n e labeled DNA from p h o t o d y n a m i e a l l y inactivated A . laidlawii. (a) Untreated unirradiated ceils. (b) Cells treated with acriflavine, as described for Fig. 3, b u t n o t irradiated. (c) Cells treated with acriflavine, irradiated w i t h visible light t o 50---60% survival, and held in holding buffer for 2 rain, as descztbed for Fig. 4. (d) Cells treated as per sample c above, e x c e p t t h a t the holding time was 20 min. (e) Cells t r e a t e d like sample d al~.ove, e x c e p t t h a t the 20 rain h o l d i n g was in tryptose b r o t h . (f) Cells treated w i t h acriflavine, irradiated w i t h visible light t o 0.4% survival and analyzed either i m m e d i a t e l y after p h o t o d y n a m i c i na c t i va t i on (o) or after 30 rain liquid holdin g (o). S e d i m e n t a t i o n was from right to left.
Effect of acriflavine on membrane permeability. It has been suggested that cell membrane damage might also be involved in photodynamic inactivation [3,6]. Mycoplasmas are useful systems for studying the role of cell envelopes in photodynamic inactivation since the envelope is only a single lipoprotein cell membrane. Therefore, to examine possible cell membrane damage during photodynamic inactivation, glucose uptake in A. laidlawii cells was studied by following the uptake of a-[3H]methyl glucose, a nonmetabolizable sugar. The kinetics of glucose uptake in cells which had been photodynamically inactivated to 10% survival was indistinguishable from unirradiated acriflavine treated cells (Fig. 7) and from untreated unirradiated cells (data n o t shown).
632 These complex kinetics are characteristic of glucose uptake in mycoplasmas and are believed to reflect the approach to a steady-state of the influx and efflux processes. Alkaline sucrose gradient analysis o f photodynamically inactivated cell DNA. DNA of photodynamically inactivated cells during liquid holding recovery was examined by analyzing cell lysates by velocity sedimentation in alkaline 5--20% (w/v) sucrose gradients. A. laidlawii cells were grown overnight in tryptose broth containing 5 gCi [SH]deoxythymidine/ml; then treated with acriflavine, photodynamically inactivated and analyzed on alkaline sucrose gradients. Unirradiated acriflavine treated cells (Fig. 8b) have the same number average single-stranded DNA molecular weight (4.7.108) as untreated unirradiated cells (Fig. 8a). After irradiation with visible light to a survival of 50--60% and 2 rain of liquid holding, the single-stranded DNA molecular weight was reduced to 2.7 • l 0 s (Fig. 8c); after 20 min of holding the single stranded DNA molecular weight had increased to 4.7 • l 0 s (Fig. 8d), the same value as in control cells. Similar recovery was observed by holding the irradiated cells for 20 min in tryptose broth instead of holding buffer (Fig. 8e). A sample of cells was irradiated to a 0.4% survival and divided into two parts: one was taken immediately for alkaline sucrose gradient analysis and the other was held for 30 min before gradient analysis. DNA from ceils analyzed just after photodynamic inactivation showed extensive degradation (Fig. 8f) and by 30 min holding time almost all of the remaining acid precipitable radioactivity remained at the top of the gradient. Discussion
The results presented here show that treatment of A. laidlawii cells with a low concentration of acriflavine (0.5 ~g/ml) has little effect on cell viability (Table I) or on the rates of DNA and protein synthesis (Fig. 1). However, exposure of acriflavine treated A. laidlawii and M. gallisepticum cells to visible light leads to photodynamic inactivation (Fig. 3). In A. laidlawii, depending on the extent of the inactivation, the damage induced by photodynamic inactivation can be repaired if the irradiated cells are incubated in the dark in buffer (Fig. 4). Analysis of the DNA of irradiated A. laidlawii cells (Fig. 8) shows that photodynamic inactivation induces strand breaks which can be repaired during liquid holding. However, cells irradiated long enough to give extensive inactivation (10% cell survival or less) are unable to recover viability and the DNA analysis shows that, in these cells, the DNA is progressively degraded during holding. To examine another possible aspect of photodynamically induced damage, in heavily irradiated A. laidlawii cells glucose uptake was determined as a measure of membrane integrity. No difference in glucose uptake was observed between control cells and photodynamically inactivated cells (Fig. 7). This indicates that the cell membrane is not significantly affected by photodynamic inactivation.
633 Acknowledgements We thank Mr. David Gerling for his technical assistance. These studies were supported by USPHS grant AI-07939 from the National Institute of Allergy and Infectious Diseases. References 1 L o c h m a n , E.R. and Micheler, A. (1973) in Physicochemical Properties of Nucleic Acid (Duchesne, J., ed.), Vol. 1, pp. 223--267, Academic Press, New Y o r k 2 Georghiou, S. (1977) P h o t o c h e m . Photobiol. 26, 59--68 3 Cramer, W.A. and Uretz, R.B. (1966) Virology 28, 142--149 4 Freifelder, D. and Uretz, R.B. (1966) Virology 30, 97--103 5 Spikes, J.D. and Livingston, R. (1969) Adv. Radiat. Biol. 3, 29--121 6 Ito, T. and Kobayashi, K. (1977) Photochem. Photobiol. 25, 399---401 7 Sastry, K.S. and Gordon, M.P. (1966) Biochim. Biophys. A c t a 129, 42--48 8 Jacob, H.E. (1971) P h o t o c h e m . Photobiol. 14, 743--745 9 Ziebell, R., I m r a y , F.P. and MacPhee, D.G. (1977) J. Gen. Microbiol. 101, 143--149 10 Smith, K.C. (1962) Biochem. Biophys. Res. C o m m u n . 8 , 1 5 7 - - 1 6 3 11 Harm, W. (1968) Biochem. Biophys. Res. Commun. 3 2 , 3 5 0 - - 3 6 8 12 Janovska, E., Zhestjanikov, J. and Vizdalova, M. (1970) Intern. J. Radiat. Biol. 1 8 , 3 1 7 - - 3 2 9 13 Uretz, R.B. (1964) Radia£. Res. 2 2 , 2 4 5 14 Das, J., Bagchi, B. and Chaudhuri, U. (1974) P h o t o c h e m . Photobiol. 1 9 , 3 1 7 - - 3 1 9 15 Harrison, A.P., Hafner, J.L. and O'Bryan, C. (1972) Mutat. Res. 14, 447--449 16 I mray , F.P. and MacPhee, D.G. (1973) Mol. Gen. Genet. 123, 289--298 17 Manfloff, J. and Morowltz, H.J. (1972) Bacteriol. Rev. 3 6 , 2 6 3 - - 2 9 0 18 Das, J., Maniloff, J. and Bhattacharjee, S.B. (1972) Biochim. Biophys. A c t a 2 6 9 , 1 8 9 - - 1 9 7 19 Ghosh, A., Das, J. and Maniloff, J. (1977) J. Mol. Biol. 1 1 6 , 3 3 7 - - 3 4 4 20 Ghosh, A., Das, J. and Manfloff, J. (1978) Biochim. Biophys. Acta 5 4 3 , 5 7 0 - - 5 7 5 21 Das, J., Nowak, J.A. and Maniloff, J. (1977) J. Bacteriol. 129, 1424--1427 22 Quinlan, D.C., Liss, A. and Maniloff, J. (1972) Microbios. 6 , 1 7 9 - - 1 8 5 23 Das, J. and Maniloff, J. (1975) Biochem. Biophys. Res. C o m m u n . 6 6 , 5 9 9 - - 6 0 5 24 R o t t e m , S. and Razin, S. (1969) J. Bacterlol. 9 7 , 7 8 7 - - 7 9 2 25 Hurwitz, J., F u r t h , J.J., Malamy, J. and Alexander, M. (1962) Proc. Natl. Acad. Sci. U.S. 48, 1 2 2 2 - 1230 26 Woese, C., Naono, S., Softer, R. and Gros, F. (1963) Biochem. Biophys. Res. C ommun. 1 1 , 4 3 5 - - 4 4 0
Biochimica et Biophysica Acta, 544 (1978) 624--633 © Elsevier/North-Holland Biomedical Press
BBA 28726
PHOTODYNAMIC INACTIVATION AND ITS REPAIR IN MYCOPLASMAS
UTPAL CHAUDHURI,JYOTIRMOY DAS and JACK MANILOFF * Department of Microbiology, University of Rochester, Medical Center, Rochester, N.Y. 14642 (U.S.A.) (Received May 8th, 1978)
Summary Photodynamic inactivation is the loss in viability observed when organic dyetreated cells are exposed to visible light and molecular oxygen. The photodynamic inactivation of mycoplasmas, the smallest free living cells, has been studied. Depending on the extent of inactivation in Acholeplasma laidlawii, photodynamic induced damage can be repaired if the irradiated cells are incubated in the dark in buffer. Analysis of the DNA of these cells shows that photodynamic inactivation induces single strand breaks which can be repaired during liquid holding. To examine possible damage to the cell membrane, glucose uptake was studied as a permeability measure. Neither acriflavine nor photodynamic inactivation had any measurable effect on membrane permeability.
Introduction Aminoacridine dyes have antibacterial and antiviral effects and can induce frameshift mutations. When acridine treated bacterial cells are exposed to visible light and molecular oxygen there is a loss of viability, referred to as photodynamic inactivation [1,2]. This inactivation is believed to be primarily due to acridine dye-DNA interactions [2], although both proteins and cell membranes have been suggested to also be involved in photodynamic inactivation [3--6]. DNA lesions are known to occur during photodynamic inactivation and three possible models for this effect have been proposed: (i) base modification [7]; (ii) single-strand breaks [8,9]; and (iii) cross-linking of DNA and pro~ins [10]. Most studies have not explicitly examined the possible repair of photodynamically induced DNA damage, which has led to some controversy as to whether this damage can be repaired [ 1 1 - 1 3 ] . Das et al. [14] investigated the * T o w h o m r e p l l n t r e q u e s t s s h o u l d be a d d r e s s e d .
625 liquid-holding repair capabilities of radiation resistant Escherichia coli B/r and radiation sensitive E. coli BB-1 strains after photodynamic damage and showed that, while the B/r strain can repair such damage, the Bs-1 strain lacks this capability. The polA and rec genes have been found to have a role in repair of photodynamic damage [9,15,16]. In recent studies the repair capabilities of the mycoplasmas have been examined. These prokaryotes are the smallest free living cells. They do not have cell walls and each cell is bounded by a single lipoprotein membrane [17]. Acholeplasma laidlawii, a mycoplasma with a genome size of 1 • 109 daltons, has been shown to have both dark (excision) and light repair mechanisms for the repair of ultraviolet light-induced DNA damage [18]. Mycoplasma gaUisepticum, a mycoplasma with a genome size of 0.5 • 109 daltons, was found to lack both of these repair mechanisms [19]. Treatment of ultraviolet light-irradiated A. laidlawii with the acridine dye acriflavine inhibits excision repair, but acriflavine treatment before irradiation partially protects the cells against inactivation [20]. Acriflavine treated A. laidlawii cells also show reduced host cell reactivation of ultraviolet light-irradiated mycoplasmaviruses [21]. We report here on studies of photodynamic inactivation and its repair in mycoplasmas. These studies show that A. laidlawii can repair some photodynamic inactivation damage to its DNA and that cell permeability is not affected. Materials and Methods
Cells, media and buffers. The mycoplasmas used in this study were Acholeplasma laidlawii strain JA1 and Mycoplasma gallisepticum strain A5969. These are the strains used in previous DNA repair studies reported from this laboratory [19--21]. A. laidlawii cells were grown in tryptose broth and assayed on tryptose agar plates as previously described [22]. M. gallisepticum cells were grown in mycoplasma broth base (MBB) medium and assayed on MBB agar plates as described previously [19]. ~-Buffer was 0.05 M trishydroxymethylaminomethane (Tris), 0.156 M NaC1 and 0.01 M ~-mercaptoethanol, pH 7.4. The holding buffer was one part growth medium and nine parts ~-buffer. Acriflavine treatment. Acrifiavine hydrochloride was obtained from Sigma Chemical Corp. (St. Louis, Mo.). Acriflavine solutions (1 mg/ml) were made in sterile distilled water and stored in the dark at 0--4°C. The acriflavine solution had an adsorption maximum at 445 nm and molar extinction coefficient of 3.4-104 M-1 at this wavelength. For acriflavine treatment, exponentially growing cells were harvested by centrifugation (8000 rev./min for 10 min) and resuspended (to a final titer of 1--2 • l 0 s colony-forming units/ml) in holding buffer, containing acriflavine at a final concentration of 0.5 pg/ml. The cell suspension was incubated in the dark for 1 h at 37°C. Irradiation o f cells. 2-ml cell suspensions, either untreated or treated for 1 h with acriflavine were put in 60-mm petri dishes and irradiated with a 40-W fluorescent lamp at a distance of 14 cm. The cell suspension was kept in an ice bath during irradiation and stirred ocasionally. At different times, 0.1-ml samples were removed and assayed for colony-forming units. All operations after
626 irradiation were carried out in dim light. Acriflavine solutions irradiated this way for 60 min showed no detectable change in absorbance at 445 nm, indicating that any bleaching of the dye was insignificant during these experiments. Liquid holding recovery. Samples of irradiated cells were diluted 100-fold in holding buffer and incubated in the dark at 37°C. At different times of incubation samples were removed and assayed for colony-forming units. Macromolecular synthesis. To measure DNA, RNA or protein synthesis, [3H]deoxythymidine (20 ~Ci/ml), [3H]uridine (20 pCi/ml) or [14C]leucine (10 gCi/ml) was added, respectively, to a cell suspension. At different times duplicate samples were removed and assayed for trichloroacetic acid precipitable radioactivity as described previously [23]. Glucose uptake. Glucose transport in acriflavine treated cells was examined following the method of Rottem and Razin [24]. For these experiments the buffer was prepared as follows: to nine parts of a buffer containing 0.05 M Tris-HC1, 0.15 M NaC1 and 0.01 M MgC12 (pH 8.0) was added one part of tryptose broth containing neither glucose nor pleuropneumonia-like organism (PPLO) serum fraction [3.0.methyl-3H]-D-Glucose (25 pCi(ml) (New England Nuclear, Boston, Mass.) was added to cell suspensions in this buffer and thecells were incubated in the dark at 37 ° C. At different times after addition of the labelled glucose, 50-pl samples were withdrawn and filtered. The filters containing cells were washed, dried and assayed for radioactivity. Sedimentation in alkaline sucrose gradients. Control and photodynamically inactivated cells were analyzed in 5 ml 5--20% (w/v) sucrose gradients (adjusted to pH 12.0 with NaOH) as described previously [18]. The sucrose was in ~-buffer containing 0.005 M EDTA. Sedimentation was in a Beckman SW50.1 rotor for 2 h at 28 000 rev./min. Calculation o f molecular weights. From the gradient analysis, number average molecular weights of cell DNA's were calculated relative to T4 marker DNA (included in all gradients), as previously described [ 18]. Survival curves. From the photodynamic inactivation data survival curves were drawn using a least-square data analysis by a computer program described previously [ 18]. Results
Effect o f acriflavine on cell growth and macromoleeular synthesis. Exponentially growing mycoplasma cells were transferred to prewarmed fresh medium containing different concentrations of acriflavine and incubated at 37°C in the dark for 3 h (a little over one doubling time). Cell growth (Table I) was not significantly affected by up to 1 pg acriflavine/ml. At increasing acrifiavine concentrations cell growth was inhibited and finally a decrease in cell viability was observed. M. gallisepticum was more sensitive to acriflavine than A. laidlawii. The synthesis of DNA, RNA and protein was examined in A. laidlawii cells treated with different concentrations of acriflavine. For these experiments, acriflavine and labelled precursors were added at zero time and macromolecular synthesis was measured as a function of time. DNA and protein synthesis were reduced by approx. 20%, relative to untreated control cells (Figs. l a and c), by
TABLE I EFFECT OF ACRIFLAVINE
ON MYCOPLASMA GROWTH
T h e i n i t i a l cell t i t e r o f A . laidlawii w a s 1 . 3 • 1 0 8 c o l o n y - f o r m i n g u n t t s / m l a n d o f M. g a l | i s e p t i c u m w a s 7.7 • 1 0 ~ c o l o n y - f o r m i n g u n i t s / m l . T h e f i n a l cell t i t e r w a s m e a s u r e d a f t e r 3 h i n c u b a t i o n a t 3 7 ° C in t h e da~k in v a r i o u s a c r i f l a v i n e c o n c e n t r a t i o n s . Acrlflavine (#g/ml)
Final titer/Initiai titer M. g a l l i s e p t i c u m
A. laidZawil 0 0.5 1.0 2.0 5.0 10.0
2.2 1.9 1.9 1.1 1.1 0.5
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Fig. 1. E f f e c t o f v a r i o u s a c r i f l a v i n e c o n c e n t r a t i o n s o n A. laidlawii m a c r o m o l e c u l a r s y n t h e s i s . A t z e r o t i m e , cells i n t r y p t o s e b r o t h w e r e m i x e d w i t h a e r i f l a v i n e a n d (a) [ 3 H ] d e o x y t h y m i d i n e , ( b ) [ 3 H ] u r i d i n e , o r (e) [ 1 4 C ] l e u e i n e , a n d t r i e h l o r o a e e t i c a e i d - p r e e i p i t a b l e r a d i o a c t i v i t y w a s m e a s u r e d as a f u n c t i o n o f i n c u b a tion thne at 37°C in the dark. The final acrlflavine concentrations were: o, no dye; A 0.5 #g/ml; a, 1 #g/ m l ; o, 2 / ~ g / m l ; a , 5 / z g / m l ; a 1 0 / ~ g / m l . F i g . 2. D N A s y n t h e s i s in A . loidIawli a f t e r r e m o v a l o f a c r i f l a v i n e . Ceils w e r e i n c u b a t e d i n t r y p t o e e b r o t h , c o n t a i n i n g (o) n o d y e , (z~) 0 . 5 , (m) 5 o r (J,) 1 0 # g a c r i f l a v i n e l m l , f o r 1 h a t 3 7 ° C in t h e d a r k . C a l l l w e r e centrifuged, washed once with trYPtose broth and resuspended in fresh tryptose broth containing 20 #Ci [ S H ] t h y m i d i n e / m l . T h e t i m e o f l a b e l a d d i t i o n w a s t a k e n as t h e z e r o t i m e p o i n t a n d i n c o r p o r a t i o n i n t o a c i d - i n s o l u b l e m a t e r i a l w a s m e a s u r e d as a f u n c t i o n o f tirae.
628
up to 1 pg acriflavine/ml. At higher dye concentrations, both DNA and protein synthesis were almost completely inhibited. In contrast, RNA synthesis was significantly affected even at low acriflavine concentrations (Fig. lb). This is consistent with observations that in the absence of light acridine dyes inhibit bacterial DNA dependent RNA polymerase [25,26]. Reversibility o f acriflavine inhibition o f DNA synthesis. A laidlawii cells were incubated in the dark for 1 h in tryptose broth containing acriflavine, then washed free of dye and resuspended in fresh medium containing [3H]deoxythymidine. The reduction in DNA synthesis observed in cells treated with a low (0.5 pg/ml) acriflavine concentration (Fig. la) was almost completely reversed when the dye was removed (Fig. 2). DNA synthesis was inhibited by 5 pg acriflavine/ml (Fig. la), but some resumption in synthesis was seen after acriflavine removal (Fig. 2). However, inhibition of DNA synthesis by 10 ug acriflavine/ml (Fig. la) was irreversible (Fig. 2). Since incubation in the dark in 0.5 pg acriflavine/ml gave only minimal effects on cell growth and macromolecular synthesis, this dye concentration was used in all subsequent photodynamic inactivation studies. Photodynamic inactivation o f mycoplasmas. These studies used both A. laidlawii and M. gallisepticum. Control experiments with untreated cells showed that the cells were not very sensitive to visible light: there was approx. 85% survival of colony-forming units for both A. laidlawii and M. gallisepticum after 40 min of irradiation (data not shown). The photodynamic inactivation curve of acriflavine-treated A. laidlawii (Fig. 3) after visible light irradiation had a shoulder after which inactivation was exponential. In terms of survival fractions, the zero dose intercept of the linear part of the A. laidlawii survival curve was 12. In contrast to the A. laidlawii curve, the M. gallisepticum survival curve was essentially exponential, with a zero dose intercept of 2 (Fig. 3). The presence of a shoulder in the photodynamic inactivation curve of A. laidlawii (Fig. 3) suggested the possible existence of a repair mechanism for photodynamic damage in these cells. Therefore, experiments were carried out to examine liquid holding recovery of such damage. Liquid holding recovery. To examine the kinetics of dark repair, A. laidlawii cells in holding buffer containing 0.5 gg acriflavine/ml were irradiated with visible light, diluted 1 : 100 with holding buffer and incubated at 37°C in the dark. Samples were removed and plated as a function of time. Untreated irradiated control cells incubated in holding buffer showed no change in titer for the holding times used in these experiments. Cells which were irradiated for short times (cell survival greater than approx. 40%) showed almost complete recovery if they were plated by 20 min of holding time (Fig. 4a). However, longer holding resulted in a decrease in recovery. Cells which were irradiated for longer times (cell survival less than 10%) showed no significant a m o u n t of liquid holding recovery (Fig. 4b) and a loss of cell titer with longer holding times. To measure the dependence of the exten~t of recovery on the initial a m o u n t of photodynamic inactivation, samples of cells were irradiated to different survival fractions, held (as above) for 20 min, and then plated. These data (Fig. 5) show that the a m o u n t of recovery is highly dependent on the initial a m o u n t of photodynamic inactivation.
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Fig. 3. P h o t o d y n a m i c i n a c t i v a t i o n o f a c r i f l a v i n e t r e a t e d A. lafdlawii (o) and M. gaUisepticum (~). Cells w e r e i n c u b a t e d i n h o l d i n g b u f f e r c o n t a i n i n g 0 . 5 ~ g a c r t f l a v t n e / m l a t 3 7 ° C f o r 1 h i n t h e d a r k . T h e cell s u s p e n s i o n w a s t h e n i r r a d i a t e d w i t h visible l i g h t a n d a s s a y e d f o r c o l o n y - f o r m i n g u n i t s as a f u n c t i o n o f irradiation time. Fig. 4 . R e p a i r k i n e t i c s o f A. laidlawii a f t e r p h o t o d y n a m i c i n a c t i v a t i o n . Cells i n h o l d i n g b u f f e t c o n t a i n l n g 0 . 5 # g a c r i f l a v t n e / m l w e r e i n a c t i v a t e d as d e s c r i b e d f o r F i g . 3 t o e i t h e r (a) 4 6 % o r Co) 4 % survival. T h e cells w e r e d i l u t e d I : I 0 0 w i t h h o l d i n g b u f f e r a n d i n c u b a t e d i n t h e d a r k a t 3 7 ° C . S a m p l e s w e r e r e m o v e d a n d a s s a y e d f o r c o l o n y - f o r m i n g u n i t s as a f u n c t i o n o f t i m e .
Macromolecular synthesis in photodynamically inactivated cells. To examine the effect of p h o t o d y n a m i c inactivation on macromolecular synthesis, A. laidlawii cells in holding buffer were treated with 0.5 ~zg acriflavine/ml for 1 h at 37°C in the dark and irradiated to various survival fractions. The cells were centrifuged, washed with tryptose broth and (at zero time) resuspended in prewarmed tryptose broth containing [3H]deoxythymidine, [3H]uridine, or [14C]leucine. Samples were removed and assayed for acid insoluble material as a function of time. In these experiments control cells were treated with acriflavine b u t n o t irradiated; hence these cells had 100% survival after the acriflavine treatment.
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Fig. 5. L i q u i d h o l d i n g r e c o v e r y o f A . ~ i d ~ w i i as a f u n c t i o n o f p h o t o d y n a m i c i n a c t i v a t i o n . Cells i n h o l d i n g b u f f e r w e r e i n a c t i v a t e d t o v a r i o u s s u r v i v a l f r a c t i o n s as d e s c r i b e d f o r Fig. 3. T h e cells w e r e t h e n d i l u t e d 1 : 100 with holding buffer, incubated for 20 rain in the dark at 37°C, and assayed for colony-forming units. Fig. 6. D N A , I%NA a n d p r o t e i n s y n t h e s i s i n p h o t o d y n a m i e a l l y i n a c t i v a t e d A . laidlawii. Cells i n h o l d i n g b u f f e r c o n t a i n i n g 0 . 5 # g a c r i f l a v i n e / m l w e r e i n a c t i v a t e d t o v a r i o u s survival f r a c t i o n s as d e s c r i b e d f o r Fig. 3: ( e ) I 0 0 , (A) 8 0 , (~) 5 0 , a n d ( o ) 7% survival. T h e cells w e r e c e n t r i f u g e d , w a s h e d w i t h t r y p t o s e b r o t h , a n d ( a t z e r o t i m e ) r e s u s p e n d e d i n t r y p t o s e b r o t h c o n t a i n i n g (a) [3 H ] d e o x y t h y m i d i n e , (b) [3 H ] u r i d i n e , o r (c) [ 1 4 C ] l e u c i n e . A c i d p r e c i p i t a b l e r a d i o a c t i v i t y w a s m e a s u r e d as a f u n c t i o n o f t i m e .
Under these conditions (Fig. 6), cells photodynamically inactivated to 70-90% survival immediately resumed DNA synthesis; however, there was a slight lag in the resumption of RNA and protein synthesis. In contrast to this, cells inactivated to 40--60% survival had a pronounced lag of approx. 2 h before DNA, RNA and protein synthesis resumed. If cells were inactivated to a greater e x t e n t (i.e., approx. 10 percent survival) no resumption of DNA, RNA or protein synthesis was observed, even after an overnight incubation in growth medium containing labeled precursors (data not shown). These biochemical results are in agreement with the viability recovery data (Fig. 4).
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Fig. 7. Glucose u p t a k e in p h o t o d y n a m l e a i l y inactivated A . |aidlawii. Cells i n h o l d i n g buffer e o n t - t n t n g 0.5 #g acriflavine/ml were either unirradiated (e) or irradiated w i t h visible light to 10 pe rc e nt survival (o). Cells were centrifuged, washed, and (at zero time) resuspended i n buffer c o n t a i n i n g ~ - [ 3 H ] m e t h y l glucose, and u p t a k e of radioactivity essayed as a f u n c t i o n of time, as described i n Materials and Methods. Fig. 8. Alkaline sucrose gradient analysis of [ 3 H ] d e o x y t h y m i d i n e labeled DNA from p h o t o d y n a m i e a l l y inactivated A . laidlawii. (a) Untreated unirradiated ceils. (b) Cells treated with acriflavine, as described for Fig. 3, b u t n o t irradiated. (c) Cells treated with acriflavine, irradiated w i t h visible light t o 50---60% survival, and held in holding buffer for 2 rain, as descztbed for Fig. 4. (d) Cells treated as per sample c above, e x c e p t t h a t the holding time was 20 min. (e) Cells t r e a t e d like sample d al~.ove, e x c e p t t h a t the 20 rain h o l d i n g was in tryptose b r o t h . (f) Cells treated w i t h acriflavine, irradiated w i t h visible light t o 0.4% survival and analyzed either i m m e d i a t e l y after p h o t o d y n a m i c i na c t i va t i on (o) or after 30 rain liquid holdin g (o). S e d i m e n t a t i o n was from right to left.
Effect of acriflavine on membrane permeability. It has been suggested that cell membrane damage might also be involved in photodynamic inactivation [3,6]. Mycoplasmas are useful systems for studying the role of cell envelopes in photodynamic inactivation since the envelope is only a single lipoprotein cell membrane. Therefore, to examine possible cell membrane damage during photodynamic inactivation, glucose uptake in A. laidlawii cells was studied by following the uptake of a-[3H]methyl glucose, a nonmetabolizable sugar. The kinetics of glucose uptake in cells which had been photodynamically inactivated to 10% survival was indistinguishable from unirradiated acriflavine treated cells (Fig. 7) and from untreated unirradiated cells (data n o t shown).
632 These complex kinetics are characteristic of glucose uptake in mycoplasmas and are believed to reflect the approach to a steady-state of the influx and efflux processes. Alkaline sucrose gradient analysis o f photodynamically inactivated cell DNA. DNA of photodynamically inactivated cells during liquid holding recovery was examined by analyzing cell lysates by velocity sedimentation in alkaline 5--20% (w/v) sucrose gradients. A. laidlawii cells were grown overnight in tryptose broth containing 5 gCi [SH]deoxythymidine/ml; then treated with acriflavine, photodynamically inactivated and analyzed on alkaline sucrose gradients. Unirradiated acriflavine treated cells (Fig. 8b) have the same number average single-stranded DNA molecular weight (4.7.108) as untreated unirradiated cells (Fig. 8a). After irradiation with visible light to a survival of 50--60% and 2 rain of liquid holding, the single-stranded DNA molecular weight was reduced to 2.7 • l 0 s (Fig. 8c); after 20 min of holding the single stranded DNA molecular weight had increased to 4.7 • l 0 s (Fig. 8d), the same value as in control cells. Similar recovery was observed by holding the irradiated cells for 20 min in tryptose broth instead of holding buffer (Fig. 8e). A sample of cells was irradiated to a 0.4% survival and divided into two parts: one was taken immediately for alkaline sucrose gradient analysis and the other was held for 30 min before gradient analysis. DNA from ceils analyzed just after photodynamic inactivation showed extensive degradation (Fig. 8f) and by 30 min holding time almost all of the remaining acid precipitable radioactivity remained at the top of the gradient. Discussion
The results presented here show that treatment of A. laidlawii cells with a low concentration of acriflavine (0.5 ~g/ml) has little effect on cell viability (Table I) or on the rates of DNA and protein synthesis (Fig. 1). However, exposure of acriflavine treated A. laidlawii and M. gallisepticum cells to visible light leads to photodynamic inactivation (Fig. 3). In A. laidlawii, depending on the extent of the inactivation, the damage induced by photodynamic inactivation can be repaired if the irradiated cells are incubated in the dark in buffer (Fig. 4). Analysis of the DNA of irradiated A. laidlawii cells (Fig. 8) shows that photodynamic inactivation induces strand breaks which can be repaired during liquid holding. However, cells irradiated long enough to give extensive inactivation (10% cell survival or less) are unable to recover viability and the DNA analysis shows that, in these cells, the DNA is progressively degraded during holding. To examine another possible aspect of photodynamically induced damage, in heavily irradiated A. laidlawii cells glucose uptake was determined as a measure of membrane integrity. No difference in glucose uptake was observed between control cells and photodynamically inactivated cells (Fig. 7). This indicates that the cell membrane is not significantly affected by photodynamic inactivation.
633 Acknowledgements We thank Mr. David Gerling for his technical assistance. These studies were supported by USPHS grant AI-07939 from the National Institute of Allergy and Infectious Diseases. References 1 L o c h m a n , E.R. and Micheler, A. (1973) in Physicochemical Properties of Nucleic Acid (Duchesne, J., ed.), Vol. 1, pp. 223--267, Academic Press, New Y o r k 2 Georghiou, S. (1977) P h o t o c h e m . Photobiol. 26, 59--68 3 Cramer, W.A. and Uretz, R.B. (1966) Virology 28, 142--149 4 Freifelder, D. and Uretz, R.B. (1966) Virology 30, 97--103 5 Spikes, J.D. and Livingston, R. (1969) Adv. Radiat. Biol. 3, 29--121 6 Ito, T. and Kobayashi, K. (1977) Photochem. Photobiol. 25, 399---401 7 Sastry, K.S. and Gordon, M.P. (1966) Biochim. Biophys. A c t a 129, 42--48 8 Jacob, H.E. (1971) P h o t o c h e m . Photobiol. 14, 743--745 9 Ziebell, R., I m r a y , F.P. and MacPhee, D.G. (1977) J. Gen. Microbiol. 101, 143--149 10 Smith, K.C. (1962) Biochem. Biophys. Res. C o m m u n . 8 , 1 5 7 - - 1 6 3 11 Harm, W. (1968) Biochem. Biophys. Res. Commun. 3 2 , 3 5 0 - - 3 6 8 12 Janovska, E., Zhestjanikov, J. and Vizdalova, M. (1970) Intern. J. Radiat. Biol. 1 8 , 3 1 7 - - 3 2 9 13 Uretz, R.B. (1964) Radia£. Res. 2 2 , 2 4 5 14 Das, J., Bagchi, B. and Chaudhuri, U. (1974) P h o t o c h e m . Photobiol. 1 9 , 3 1 7 - - 3 1 9 15 Harrison, A.P., Hafner, J.L. and O'Bryan, C. (1972) Mutat. Res. 14, 447--449 16 I mray , F.P. and MacPhee, D.G. (1973) Mol. Gen. Genet. 123, 289--298 17 Manfloff, J. and Morowltz, H.J. (1972) Bacteriol. Rev. 3 6 , 2 6 3 - - 2 9 0 18 Das, J., Maniloff, J. and Bhattacharjee, S.B. (1972) Biochim. Biophys. A c t a 2 6 9 , 1 8 9 - - 1 9 7 19 Ghosh, A., Das, J. and Maniloff, J. (1977) J. Mol. Biol. 1 1 6 , 3 3 7 - - 3 4 4 20 Ghosh, A., Das, J. and Manfloff, J. (1978) Biochim. Biophys. Acta 5 4 3 , 5 7 0 - - 5 7 5 21 Das, J., Nowak, J.A. and Maniloff, J. (1977) J. Bacteriol. 129, 1424--1427 22 Quinlan, D.C., Liss, A. and Maniloff, J. (1972) Microbios. 6 , 1 7 9 - - 1 8 5 23 Das, J. and Maniloff, J. (1975) Biochem. Biophys. Res. C o m m u n . 6 6 , 5 9 9 - - 6 0 5 24 R o t t e m , S. and Razin, S. (1969) J. Bacterlol. 9 7 , 7 8 7 - - 7 9 2 25 Hurwitz, J., F u r t h , J.J., Malamy, J. and Alexander, M. (1962) Proc. Natl. Acad. Sci. U.S. 48, 1 2 2 2 - 1230 26 Woese, C., Naono, S., Softer, R. and Gros, F. (1963) Biochem. Biophys. Res. C ommun. 1 1 , 4 3 5 - - 4 4 0
