effects of acridine plus near ultraviolet light on escherichia coli membranes and dna in vivo

Phorochernisrry and Phoiobiology. Vol. 32, pp. 771 to 119 0 Pergamon h a s Ltd 1980. Printed in Great Britain

003 I -8655/80/120I-O77l SO2.00jO

EFFECTS OF ACRIDINE PLUS NEAR ULTRAVIOLET LIGHT ON E S C H E R I C H I A COLI MEMBRANES A N D D N A I N V I V O

STEPHEN WAGNER, WILLIAM D. TAYLOR, ALEC KEITH and WALLACE SNIPES*

Biophysics Laboratory, The Pennsylvsnin State University, Lrniversity Park, PA 16802, USA

(Received 2 May 1980; accepted 20 June 1980)

Abstract-Results from a variety of experiments indicate that photodynamic damage to E. coli treated with the hydrophobic photosensitizer acridine plus near-UV light involves both cell membranes and DNA. Split-dose survival experiments with various E. coli mutants reveal that cells defective in recA, uurA, or polA functions are all capable of recovery from photodynamic damage. Alkaline sucrose gradient analysis of DNA from control and treated cells revealed that acridine plus near-UV light treatment converts normal DNA into a more slowly sedimenting form. However, the normal DNA sedimentation properties are not restored under conditions where split-dose recovery is effective. Several lines of evidence suggest that membrane damage may be important in the inactivation of cells by acridine plus near-UV light. These include (a) a strong dependence of sensitivity on the fatty acid composition of the membranes; (b) a strong dependence of sensitivity on the osmolarity of the external medium; and (c) the extreme sensitivity of an E. coli mutant having a defect in its outer membrane barrier properties. Direct evidence that acridine plus near-UV light damages cell membranes was pro- vided by the observations that (a) the plasma membrane becomes permeable to o-nitrophenyl-p-D- galactopyranoside and (b) the outer membrane becomes permeable to lysozyme after treatment. A notable result was that cells previously sensitized to lysozyme by exposure to acridine plus near-UV light lose that sensitivity upon subsequent incubation. This strongly suggests that E. coli cells are capable of repairing damage localized in the outer membrane.

INTRODUCTION

Damage to a wide range of targets may be responsible for the observed biological effects of photodynamic action on cells (Spikes and Livingston, 1969; Berg, 1967; Goldstein and Harber, 1972; Anderson and Krinsky, 1973; Nasim and Brychcy, 1979). Some sel- ectivity for the primary site of damage may be achieved through the use of photosensitizing dyes with chemical and physical properties that restrict them to certain locations in the biological system of interest. If damage is mediated through singlet oxygen, the diffusion coefficient ( - 2.5 x cm’js) (Weast, 1976) and relatively short half-life (2 ps) (Mer- kel et a/., 1972) for this species insure that the major- ity of the damage will occur at a distance of less than loo0 8, from the absorbing dye molecules. Thus, it is not surprising that DNA is a primary target when intercalating dyes such as proflavine (De Mars, 1953; Zampieri and Greenburg, 1965; Georghiou, 1977) and methylene blue (Simon and Van Vunakis, 1962; Webb et al., 1979) are utilized.

Investigators have employed two general approaches to localize photodynamic action to membranes. One approach is to use charged, water-soluble dyes that are excluded from cells. Ito and Kobayashi have demonstrated that the photosensitizing dye toluidine blue 0 is impermeable to yeast cell mem-

*To whom all correspondence should be addressed.

branes, and have observed that its photodynamic action is essentially non-mutagenic at light doses which significantly reduce cell survival (Kobayashi and Ito, 1976; Ito and Kobayashi, 1977). Strong evidence was presented that the cell plasma membrane was a main site of damage in this system. However, a significant limitation to the use of impermeable dyes to localize damage to membranes is that, as damage accumulates, the membrane may lose its selective permeability properties and the dye may gain access to other components inside the cell.

Another approach is to employ a hydrophobic photosensitizing dye that will strongly partition into membrane structures. While such a dye is not excluded from the interior of cells, its concentration may be many times greater in membranes than in the surrounding aqueous regions. For complex eucaryotic cells damage would be expected to occur in a variety of membranes, including the cytoplasmic, mitochon- drial, reticular, and nuclear membranes. For simple procaryotic cells lacking internal membrane structures, damage may be restricted to a single membrane for gram-positive bacteria or to the inner and outer membranes of gram-negative bacteria.

In a previous study, we presented data which suggest that the hydrophobic photosensitizer acridine (see Fig. 1) plus near-UV light inactivates lipid-containing viruses via singlet oxygen-mediated damage to viral membranes (Snipes et a/., 1979). Acridine, which should not be confused with its charged, water-solu-

772 S'IZPHEN WAGNER et af.

Figure 1. Structure of acridine.

ble derivatives, has a partition coefficient of approximately 2700 between decanol and water at room temperature. Thus, while it can readily cross cell membranes, it is expected to localize strongly in the hydrocarbon zones of phospholipid bilayers. In the work presented here, we have investigated the effects of acridine and near-UV light on E . coli in an effort to determine whether cell membranes may in fact be sel- ectively damaged with this agent. Our results show (a) that both membranes and DNA are affected by acridine plus near-UV light, (b) that cells can repair the damage caused by this treatment, and (c) that the recovery is probably due to repair of damage localized in the membrane.

MATERIALS AND METHODS

Cells. nietlia and pfares. Cell strains employed in this study and their relevant characteristics are listed in Table 1. Unless otherwise indicated, all strains were grown in minimal Al glucose medium (Person and Bockrath, 1964) supplemented with 25 pg/m/ of the required amino acids and 5 pg/m/ thiamine. E. coli W3110 was supplemented with 25 pg/m/ thymidine. K1060 was grown in TBM21, a chemically defined but enriched medium previously described (Wanda er al.. 1976). supplemented with 100 pg/m/ of an unsaturated fatty acid and 200pg/m/ Triton X-100. E. coli P3478 was grown in enriched V medium (Wanda et

a/., 1976), and WU36-10 was grown in either Al or a low osmolarity minimal medium, LOMM (Munro and Bell, 1973).

All strains were assayed for colony-forming units (cfu) on plates containing V medium hardened with 1.5% (w/v) Bac- to-agar. For K1060 the plates were supplemented with 250 pg/m/ TWEEN 80.

Treatment with acridine and near-UV light. Cells were grown to approximately 1-2 x lo8 cfu/m/ at 37"C, centrifuged, and resuspended in an equal volume of Al medium lacking specific growth requirements. A stock solution of acridine at 2.5 mg/m/ in 95% ethanol was diluted 100-fold into the cell suspension. Samples were continuously bubbled with air and exposed to near-UV light from Westing- house FlSTI/BLB (black light blue) bulbs at a distance of 6.5cm. Four bulbs were arranged vertically with the sample at their geometric center. Samples were irradiated at room temperature (-22"C), and direct measure of sample temperature before and after exposure in control experiments showed less than 2'C increase for the exposure times used.

Alkaline sucrose gradients. E. coli W3110 was grown in the presence of 15 pg/m/ thymidine and 60 pCi/m/ C3H]-thymidine. Cells were centrifuged, washed, and grown for 30min in 25pg/m/ thymidine to deplete C3H]-thymidine pools. Following photodynamic treat- ments, cells suspended in Al medium were incubated in the presence of 1 mg/m/ lysozyme and 10 mM EDTA (Guthrie and Sinsheimer, 1960) for 30 min at 37°C. Approximately 5 x lo6 cells were then introduced on top of a lysing solution layered above a 5520% alkaline (pH 11) sucrose gradient (McGrath and Williams, 1966). The lysing solution consisted of 0.5% sarcosyl and 0.5 M NaOH in dis- tilled water. Samples were centrifuged at 30000rpm at 20°C for 85 min in a Beckman Model SW50.1 rotor. Frac- tions were collected and analyzed for radioactivity in a Beckman Model LS-150 scintillation counter.

Sources of materials. All materials were obtained from commercial suppliers : 0-nitrophenyl-P-D-galactopyranoside (ONPG) and isopropyl-8-D-thiogalactopyranoside (IPTG) from Calbiochem Company; oleic acid, linoleic acid, mito-

Table I . Strains of E. coli utilized and their relevant characteristics

Relevant genotype Strain Parent or phenotype Source

AB1157

AB1886 AB2463 W3110 ( t h y - ) P3478

W U36-10

K I060

RPl00

SWWSl

AB1133 (K12)

AB1157 AB1157 W2637 (K12) W3110 ( r h j . - )

W U36(B/r)

KlOOl(K12)

derivative of C600

RPlOO

rhr-1

rhi-1 [a] lacy-1

proA2 his-4 argE3

rhyA36 rh!.A36 polAl leu

[a] i w A h [a] recA13

ryr- .fahB j k l leu-6

leu-6 1 A( lac-pro)XI I I

mitomycin C-sensitive* leu-6

mitomycin C-resistant A( lac-pro)Xl I I

Bachmann (1972)

Bachmann (1972) Bachmann (1972) Bachmann (1972) Bachmann (1972)

Person et a/. (1974)

Jeffrey Sands (Lehigh University) Ronald Porter (The Pennsylvania State University) This laboratory

*Sensitivity to mitomycin C mapped between the origin of lfr6 and Ilfr Cavalli by conjugation.

Photodynamic effects on E. coli membranes and DNA 773

1 I I I I

0 Air 0 Nitrogen 0 Washed

5 10 15 20 25 Min. of Irradiation

Figure 2. Synergistic effect of acridine, near-UV light, and oxygen on W3110 survival. Squares represent cells that were treated with acridine and then washed prior to ex-

posure to near-UV light.

mycin C and Triton X-100 from Sigma Chemical Com- pany; elaidic acid from The Hormel Institute; TWEEN 80 and lysozyme from Nutritional Biochemical Company; thiamine from Schwartz-Mann Chemical Company; and acridine from Aldrich Chemical Company. C3H]-thymidine (specific activity 55.2 Ci/mmol) was purchased from New England Nuclear Corporation.

RESULTS

Inactivation of E. coli with acridine plus near-UV light

We have found that several strains of E. coli are sensitive to the synergistic effects of acridine and near-UV light in the presence of oxygen. Figure 2 shows a survival curve for W3110 in the presence (open circles) and absence (closed circles) of air. Con- trol experiments established that there is no significant inactivation of cells exposed to acridine alone or to near-UV light alone over the time periods used here. The survival curve in Fig. 2 for cells bubbled with air during irradiation exhibits a very pronounced shoulder, followed by an exponential decline in cell viability. This suggests that considerable damage must be accumulated by the cell before i t is killed, and/or that repair processes aid in recovery from the damage (Zimmer, 1961 ; Elkind and Whitmore, 1967).

An experiment was carried out to determine

whether the photosensitization by acridine is reversible. Cells exposed to acridine in the dark for 25 min were pelleted, resuspended in medium, and then expesed to near-UV light. For irradiation times up to 25 min, no decrease in survival was detected (Fig. 2, open squares). These data indicate that there is no strong binding of acridine to cell components giving rise to irreversible photosensitization.

Split-dose experiments

Split-dose survival curves were carried out to determine the possibility that cells may repair or recover from sublethal damage due to acridine plus near-UV light. Cells were exposed initially for a time sufficient to reach the exponential portion of the dose-response curve. Subsequently, one portion of the culture was maintained on ice while the other portion was incubated under growth conditions at 37°C. The incubated portion was then placed on ice for 5 min, after which both portions were centrifuged, resuspended in medium containing acridine, and further exposed to near UV light. Figure 3 gives results for several strains of E. coli treated in this manner. In all cases where the incubation period was 30 min or greater, a significant split-dose recovery was observed. While incubation for 15min was insufficient for maximum split-dose recovery (Fig. 3c), incubation for 45 min was not significantly more effective than incubation for 30min. It is worthy of notice that treated cells do not undergo cell division during the recovery period, even when incubation is as long as 45min (Fig. 3e), indicating that sublethal damage due to acridine plus near-UV light causes a substantial division delay.

The potential involvement of known DNA repair systems in E. coli was studied by carrying out split- dose survival experiments on mutant strains that are known to be defective in certain repair functions. AB1886 and AB2463 are uvrA and recA mutants, respectively, of AB1157, while P3478 is a polA mutant of W3110. All these repair-deficient mutants exhibited split-dose recovery comparable to that of their wild- type parent cells.

Sedimentation properties of treated DNA

Direct evidence that treatment of E. coli with acridine plus near-UV light has an effect on cellular DNA was obtained by analyzing the DNA of treated and control cells on alkaline sucrose gradients. These data are presented in Fig. 4. We observed that the exposure of cells to acridine alone, which does not reduce cell survival, caused an appreciable reduction in the sedimentation velocity of DNA for cells lysed immediately following a 30min exposure to the dye (Fig. 4a). This effect was reversible. however. When cells previously exposed to acridine were washed re- peatedly and then lysed, the normal DNA sedimentation pattern was restored. These data indicate that acridine is capable of reversibly binding to the cell in such a way that, upon treatment of these cells on

174

I I 1 I I I 1

0 10 20 30'"

STEPHEN WAGNER et al.

I 1 1 ' 1 I I I I I I

5 10 15 20 0 5 10 15 20

alkaline sucrose gradients, their DNA sedimentation properties are modified.

For cells treated with acridine plus near-UV light, the DNA sedimentation is reduced even further. In this case, however, washing the cells after treatment does not restore the normal sedimentation characteristics for the DNA (Fig. 4b). In order to determine whether the recovery of cells in split-dose experiments might be associated with a recovery of normal DNA sedimentation properties during the incubation period, we treated cells with acridine plus near-UV light and then incubated samples for varying periods of time under growth conditions prior to lysing on the gradients. Within the period of 3 M 5 min required for split-dose recovery, no return to normal DNA sedimentation properties was found (Fig. 4c). Even for incubation times as long as 180 min, only a slight shift of the slowly sedimenting peak toward the untreated peak was observed (data not shown). These results suggest that the sublethal damage to cells, from which they are capable of recovery, is not likely to be associated with the reduced sedimentation properties of cellular DNA observed in these experiments.

Sensitivity of K l060 grown in various unsaturated fatty acids

The experiments described in the remaining sections were all designed to stddy the effects of acridine plus near-UV light on membranes and membrane-related responses. In this section data are presented for the survival of K1060 (Bayer et al.,

1977), a fatty acid auxotroph which can neither syn- thesize nor degrade unsaturated fatty acids (Overath and Raufuss, 1967; Vagelos and Sibert, 1967), to acridine plus near-UV light. This strain of E. coli incorporates into its membrane those unsaturated fatty acids that are supplied in the growth medium, and the physical properties of the membranes then reflect the properties of the fatty acids that are utilized (Liden et a/ . , 1973). Cells were grown in the presence of either elaidic (1 8 : 1 A9 trans), oleic (1 8 : 1 A9 cis) or linoleic (18:2 A 9 * I 2 cis,cis) acid, centrifuged, and resuspended in medium free of fatty acids for exposure to acridine plus near-UV light. The survival curves are shown in Fig. 5. The resistance to treatment for cells grown in linoleic acid is very striking. This fatty acid has a melting point of -5T, and membranes containing linoleic acid are expected and have been found to be highly disordered with a high degree of molecu- lar mobility for the fatty acyl chains. Oleic and elaidic acids have melting points of 13 and 4 5 T , respectively. and cells grown on these fatty acids show sensitivities considerably greater than those grown on linoleic acid. The order of sensitivity. elaidic > oleic > linoleic, is the same as that for the rigidity of membranes containing these unsaturated fatty acids. These data strongly suggest the involvement of cell membranes in the inactivation of K1060 by acridine plus near-UV light.

Osmolarity effects

Membrane-associated processes in cells are often


Fraction Number

Figure 4. Alkaline sucrose gradients of W3110 DNA. Frac- tions were collected from the bottom of SW50.1 centrifuge tubes. Open circles designate control W3110 DNA. (a) Closed circles represent cells pretreated with acridine. Open squares indicate cells pretreated with acridine and subsequently washed on a millipore filter. (b) Open squares designate cells treated with acridine and 10 min exposure to near-UV light. Closed squares represent acridine plus near-UV light treated cells followed by washing on a millipore filter. (c) Closed circles indicate cells treated with acridine and 10 min near-UV light. Closed squares designate acridine plus near-UV light treated cells followed by a 45 min incubation under aerobic growth conditions at

37°C.

influenced by the osmolarity of the surrounding culture medium. The results of a set of experiments designed to determine the effects of external osmolarity on sensitivity to acridine plus near-UV light are presented in Fig. 6. These data show that cells are- most sensitive to acridine plus near-UV light when grown and treated in LOMM, a low osmolarity minimal medium (Munro and Bell, 1973). Increasing osmotic strength in going from LOMM to Al medium and then to Al with 250mM NaCI progressively reduced the sensitivity of cells to treatment. These results are consistent with membrane involvement in PAP. 3 2 1 6 ~

the inactivation of cells by acridine plus near-UV light.

Effects of acridine plus near-UV light on the permeability of the plasma membrane

A direct assay for damage to the plasma membrane upon treatment with acridine plus near-UV light was developed, based on loss of the permeability barrier to the 8-galactosidase indicator, o-nitrophenyl-P-D- galactopyranoside (ONPG). E. coli ABll57 has a non-revertible lac-Y mutation and is therefore unable to transport P-galactosides (Bachmann el al., 1967; Bachmann, 1972). T iese cells can, however, be induced to produce large quantities of 8-galactosidase by growth in the presence of isopropylthiogalactopyr- anoside (IPTG), which can passively diffuse through the plasma membrane. Once induced, however, these intact cells cannot cleave externally supplied ONPG. since this molecule cannot cross the membrane of the permease-deficient cells. Thus, for our purposes, the hydrolysis of ONPG by cells induced with IPTG can be employed as a method to measure loss of the selective barrier properties of the plasma membrane.

The experiment was conducted as follows. Cells grown in Al medium with glycerol (4g//) replacing glucose as carbon source were induced with IPTG (1 mM) for 100min. The culture was centrifuged. resuspended at 108cfu/m/ in medium lacking the required amino acids, and divided into four portions. One sample was treated with acridine plus 10min near-UV light, another with acridine alone, another with light alone, and a fourth maintained as an untreated control. To all four samples ONPG was added to a final concentration of 5 mM. The samples were incubated at 37°C for 7.5 min, centrifuged, and the absorbance of the supernatant measured at 420nm. Results are shown in Table 2. The only sample showing a significant increase in absorbance is the one treated with acridine plus near-UV light. Ad- ditional control experiments showed that P-galactosidase does not leak out of treated cells, since the supernatant from a treated sample gave no hydrolysis of ONPG (data not shown). These data indicate that acridine plus near-UV light damages the plasma membrane of AB1157 in such a way that ONPG can leak into the cell and the cleavage product o-nitrophe- no1 can leak out.

Sensitivity of RP100 and i t s miromjrin C-resistant revertant to acridine plus light

E . coli and many other gram-negative organisms are insensitive to several dyes and drugs at doses that are toxic to gram-positive organisms, presumably because these agents do not readily permeate the gram-negative outer membrane. Some examples of these agents are charged, water-soluble, photosensitizing dyes such as methylene blue, eosin Y , and acridine orange, and drugs such as mitomycin C and phen- ethyl alcohol. E. coli N43. a mutant sensitive to the above compounds, also has increased sensitivity to

776 STEPHEN WAGNER et a/.

c 0

e - V

LL

I I I

linoleic 0 oleic 0 elaidic

I I I 10 20 30

Min of Irradiation

Figure. 5. The effect of fatty acid supplementation of the sensitivity of K1060 to acridine plus near-UV light.

the detergent sodium dodecyl sulfate. It was therefore hypothesized that this mutation, designated acrA, gives rise to a deficiency in a protein necessary for the structural integrity of the outer membrane (Naka- mura. 1964, 1968).

RPlOO is a mitomycin C-sensitive derivative of C6OO into which acrA was introduced by conjugation (R. Porter, Microbiology Program, The Pennsylvania State University; personal communication). In order to see if this mutation conferred acridine plus light sensitivity, we carried out dose-response experiments on RPlOO and a spontaneous mitomycin C-resistant revertant, designated SWWSl. SWWS! was isolated on an enriched medium plate containing 0.5 pg/m/ mitomycin C. The acridine plus light survival curves of RPlOO and SWWSl are presented in Fig. 7. RPlOO is extremely sensitive to acridine plus light, while SWWSl displays typical wild-type sensitivity. These data lend support to the notion that the outer membrane may be an important site for damage induced by acridine plus light.

Assay for outer membrane damage

This final section describes an assay for outer membrane damage based on the loss of the ability of the outer membrane to exclude lysozyme. In addition. some data are presented which indicate that cells can

repair damage to their outer membrane. E. coli is normally insensitive to lysozyme unless the outer membrane is damaged by some treatment, such as divalent ion depletion with EDTA. Our experiments were designed to determine whether sublethal doses of acridine plus near-UV light can render cells sensitive to inactivation upon subsequent exposure to lysozyme, and whether treated cells can recover from this sensitivity. W3110 was first exposed to acridine plus near-UV light and plated for survivors. The treated culture was divided into two portions, one of which was exposed to lysozyme (1 mg/m/) for 30 min at 37°C and then plated for survivors. The other portion was incubated under growth conditions for 45 min, plated, exposed to lysozyme, and again plated for cfu. The results of this experiment are summarized in Table 3.

A comparison of samples 2 and 3 shows that cells treated with acridine plus near-UV light become sensitive to inactivation by lysozyme, suggesting that this treatment damages the outer membrane. Control experiments showed that cells treated with acridine alone or light alone are not affected by exposure to lysozyme (data not shown). Furthermore, a comparison of samples 4 and 5 shows that previously treated cells lose their sensitivity to lysozyme during a 45-min incubation under growth conditions. These results strongly suggest that E . coli cells can repair damaged


Min. of lrrodiotion

0 A l t NoCl

0 LOMM

I I I 2 4 6

0 A l t NoCl A Al 0 LOMM

Figure 6. The effect of external osmolarity on WU36-10 survival to acridine plus near-UV light treatment. Cells were grown and subsequently irradiated in Al medium, Al medium containing 250 mM NaCI, or LOMM (Munro and

Bell, 1973).

outer membranes and regain the capacity to exclude lysozyme.

DISCUSSION

Results from a great variety of experiments have firmly established that damaged DNA may be repaired in many organisms. In E. coli there are several different repair mechanisms, including photo- reactivation. dark repair, and the inducible error-prone repair systems (Hanawalt et al., 1979). For the meta- bolic repair pathways, split-dose recovery is an indi- cation that the cells which have accumulated sub-

Table 2. Effects of acridine plus near-UV light on the permeability of E . coli

AB1157 cells to ONPG

Absorbance* Treatment (420 nm)

Control 0.04 Acridine 0.07 Near-UV 0.06 Acridine + near-UV 0.37

*Measured in supernatant after 7.5 min incubation at 37'C.

lethal amounts of damage can repair that damage during incubation between exposures (Elkind and Sutton, 1960). If the repair pathway is non-functional, e.g. in repair-deficient mutants, the recovery is strongly impaired.

We observed that the survival curves for E. coli exposed to acridine plus near-UV light exhibit a pronounced shoulder, and that a very effective split-dose recovery occurs for incubation periods of 30min or greater. This split-dose recovery was effective even in mutants defective in the recA, uvrA, and polA enzyme repair functions. Other experiments carried out in our laboratory but not reported here have shown that the error-prone repair pathway is not induced to any significant extent by acridine plus near-UV light under the conditions of our experiments. Thus, while it is evident that repair of some type is taking place, it cannot be attributed in any direct and straightforward manner to the known DNA repair processes in E. coli. Although the mutants studied are, in some cases, considerably more sensitive to acridine plus near-UV than are their parent strains, the survivors of an initial exposure still recover with good efficiency during incubation.

The analysis of DNA from treated cells and untreated controls on alkaline sucrose gradients established that there is significant DNA damage for the exposures used in the cell survival experiments.

I I I I 0 5 10 15 2c

M i n of lrrodiotion

Figure 7. Sensitivity of RPIOO and its mitomycin C-resistant revertant to acridine plus light treatment.

778 STEPHEN WAGNER e t al.

Table 3. Effects of acridine plus near-UV light and subsequent incubation on the sensitivity of E . coli W3110 to lysozyme

Sample Treatment ‘lo survival

1 Control 100 2 Acridine + near-UV 0.87 & 0.13 3 Acridine + near-UV + lysozyme 0.13 +_ 0.03 4 Acridine + near-UV + incubation* 0.62 0.10 5 0.61 & 0.09 Acridine + near-UV + incubation* + lysozyme

*Cells were incubated for 45min at thymidine.

These results show that acridine plus near-UV light produces either single-strand breaks, double-strand breaks, alkali-labile bonds, or some combination of these types of damage. However, this damage to DNA is not repaired under conditions that allow damaged cells to recover from sublethal exposures.

Modification of the lipid fatty acid composition in strain K1060 resulted in very large changes in sensitivity to acridine plus near-UV light, implicating membranes as a likely site for damage. There are several factors of potential significance to these results. First, the extent of partitioning of acridine into the lipid hydrocarbon zones may depend on the fatty acid composition of the phospholipids. However, hydrophobic impurity molecules generally partition more favorably into more fluid membranes (Tinberg et al., 1972), so that cells supplemented with linoleic acid should be the most sensitive of those studied. This is contrary to the experimental results. Second, if lipid oxidation reactions were associated with cell death, the extent of these reactions should be greatest for cells supplemented with linoleic acid, since lipid oxidations occur at double-bond sites (Foote, 1968) and are especially prevalent for polyunsaturates. This again is contrary to the data. Third, if protein oxidation reactions in the membrane were responsible for the damage. this might be reduced in cells supplemented with linoleic acid, since this compound might compete more effectively with proteins for the oxidiz- ing species. This effect would be consistent with increased sdrvival in cells supplemented with linoleic acid. Finally, the physical state of the membrane as influenced by its fatty acid composition could have some direct or indirect effect on the ability of the cell

37‘C in Al medium supplemented with

to survive in the presence of damage from acridine plus near-UV light.

Additional evidence implicating the involvement of cell membranes was obtained in the study of the effects of osmolarity on survival. Likewise, the extreme sensitivity of strain RP100, which has defective outer membrane barrier properties, is suggestive of an effect of acridine plus near-UV light on membranes. Direct evidence for membrane damage comes from the increased permeability of the plasma membrane to ONPG and the outer membrane to lysozyme. While these latter data cannot be correlated with cell inactivation by the treatment, they establish with a high degree of certainty that acridine plus near-UV light does damage E . coli membranes under conditions where cell survival is diminished.

An observation of particular interest was that cells sensitized to lysozyme by treatment with acridine plus near-UV light can lose their sensitivity during incubation. We interpret this result as evidence for outer membrane repair. The time required for recovery from lysozyme sensitivity is comparable to that for split-dose recovery of survival. Jacob and Hamann (1975) have reported a form of recovery from osmotic fragility in Proteus mirabilis cells treated with methylene blue, but in that case recovery was complete within 1-2 min of incubation. Experiments currently underway in our laboratory are designed to detect and characterize the repair of damage to the plasma membranes of cells.

Acknowledgemenr-This work was supported by a grant from the US Department of Energy.

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