Photochern. Phorobiol. Vol. 36, pp. 255 to 258, 1982 Printed in Great Britain. All rights reserved

RESEARCH NOTE

003 1 -8655/82/080255-04$03.00/0 Copyright 0 1982 Pergamon Press Ltd

EFFECTS OF ACRIDINE PLUS NEAR-ULTRAVIOLET LIGHT O N THE OUTER MEMBRANE OF

E S C H E R I C H I A COLI

STEPHEN WAGNER and WALLACE SNIPES* Biophysics Laboratory, The Pennsylvania State University, University Park,

PA 16802, USA

(Received 23 Nouember 1981; accepred 10 March 1982)

Abstract-The hydrophobic photosensitizer acridine plus near-ultraviolet light damages both plasma membranes and outer membranes in Escherichia coli. Two lines of evidence are presented that outer membrane proteins are affected by acridine plus near-ultraviolet light treatment and that the effect is selective for certain proteins. First, analysis of outer membrane proteins on sodium dodecylsulfate polyacrylamide gels revealed that some protein bands are diminished upon treatment while others remain unaltered. New bands also appear, suggesting degradation or crosslinking reactions. Second, bacteriophage adsorption studies showed that treatment of E. coli F cells with acridine plus near- ultraviolet light causes a loss in functionality of the receptor for phage T5. Treatment of E. coli AB1157 cells under comparable conditions has no discernable effect on the functionality of the receptor for phage BF23.

INTRODUCTION

In previous studies we have presented evidence that the hydrophobic photosensitizer acridine plus near ultraviolet (near-UV)? light damages both the plasma membrane and the outer membrane of Escherichia coli (Wagner et al., 1980, 1982). Experimental results that support plasma membrane damage included an increased permeability of treated cells to o-nitrophe- nyl-B-D-galactopyranoside and an increased sensi- tivity of treated cells t o lethal osmotic shock. Direct evidence for outer membrane damage came from ex- periments in which treated cells become sensitive to lysozyme, an enzyme that normally cannot penetrate the outer membrane of E. coli . Although these results establish the loss of membrane integrity in cells treated with acridine plus near-UV light, little evi- dence is available regarding what membrane com- ponents are damaged by this treatment or the physico- chemical nature of this damage.

The present study provides two lines of evidence that outer membrane proteins are affected by treat- ment with acridine plus near-UV light. First, sodium dodecylsulfate (SDS) polyacrylamide gel electro- phoresis revealed a selective loss of certain outer membrane proteins from treated cells as well as the

*To whom correspondence and reprints should be addressed.

TAbbreuiarions: c.f.u., colony-forming units; m.o.i., multi- plicity of infection; near-UV, near ultraviolet; p.f.u., pla- que-forming units; SDS, sodium dodecylsulfate; wt/vol, weight per volume.

appearance of modified protein species. Second, bac- teriophage adsorption studies showed that the recep- tor for phage T5 loses its functionality upon treat- ment, whereas the receptor for phage BF23 remains fully functional after treatment under comparable conditions. These results establish a degree of speci- ficity in the action of acridine plus near-UV light on the components of E . coli outer membranes.

MATERIALS AND METHODS

The two bacterial strains used in this study were E . coli ABll57, obtained from Dr. Barbara Bachmann and E. coli F, obtained from Dr. James McCorquodale. Bacteriophage T5 and BF23 were gifts from Dr. McCorquodale. Cells were grown in V medium (Wanda et al., 1976) to approx. 2 x lo8 colony-forming units (c.f.u.)/ml, centrifuged, and resuspended in A1 medium (Person and Bockrath, 1964) for exposure to acridine plus near-UV light. The apparatus and procedure for this treatment have been previously de- scribed (Wagner et al., 1980). For phage adsorption studies, T5 and BF23 were assayed for plaque-forming units (p.f.u.) on E. coli F and E . coli AB1157, respectively, by the agar overlay technique. Plates contained V medium hardened with 1.5% (wtjvol) Bacto agar. Top agar consisted of V medium lacking yeast extract, hardened with 0.5% (wt/vol) Bacto agar.

The 1251 surface-labeling of bacterial cells was carried out as follows. A 25-me culture of cells was grown in V medium to 2 x 10' c.f.u./ml, centrifuged and resuspended in 500 jd of phosphate-buffered saline containing 5 mM glucose and 400pCijml 1251 in the form of sodium iodide. To the sample was added 5 O p f of an enzyme sol- ution containing 1 mg/mC lactoperoxidase and 5 units/mf glucose oxidase. The sample was incubated at room tem- perature and vortexed periodically to ensure proper aera- tion. Following incubation, 5 mY of A1 medium was added to the reaction mixture. The labeled cells were centrifuged

255

256 STEPHEN WAGNER and WALLACE SNPES

and washed twice in A1 medium to remove lactoperoxi- dase, glucose oxidase, and unincorporated 1251. Acridine was added to a final concentration of 25 pg/m/, after which the cells were exposed to near-UV light and concentrated by centrifugation for electrophoresis.

Sodium dodecylsulfate polyacrylamide gel electro- phoresis was carried out on '"I surface-labeled cells by the method of Laemmli (1970). The sample solution con- tained 2 g of SDS, 0.61 g of Tris-HC1, 5 me of b-mercap- toethanol and 5mg of bromophenol blue, with the final volume brought to 100ml distilled water (pH 7.0). Ap- proximately 1 5 0 d of sample and sample solution were mixed and heated to 100°C for 5min. The resulting sol- ution was then introduced onto a 4% polyacrylamide stacking gel, under which was a 12% separating gel. Samples were electrophoresed for approx. 6 h under a con- stant current of 20 mA. After electrophoresis, the gels were fixed in a 10% acetic acid and 30% methanol solution for 1 h. Gels were subsequently incubated with 100m/ of EN'HANCE for 1 h and then allowed to swell in distilled water for 1 h. This fluorography technique was initially developed by Laskey and Mills (1975). The gels were then dried and incubated with X-ray film in the dark for 5 days at -40°C. Prior to incubation, the X-ray film was exposed to low intensity light to ensure a linear relationship between the amount of radioactivity and the number of exposed grains (Laskey and Mills, 1975).

All materials utilized were obtained from commercial suppliers: '''I and EN3HANCE from New England Nuclear Corp., lactoperoxidase from Calbiochem Corp., and glucose oxidase from Sigma Chemical Co.

RESULTS

Analysis of outer membrane proteins The enzyme lactoperoxidase has been used in many

investigations to selectively surface-label the mem- branes of cells with '''I (Arntzen et al., 1974; Hogg, 1974; Haustein, 1975). Since lactoperoxidase cannot enter cells, the reactive ' 2 5 I species produced primar- ily labels exposed tyrosine residues (Bayse et al., 1972) on cell-surface proteins. We utilized this technique to label the outer membrane proteins of E. coli cells and analyzed these proteins in cells treated with acridine plus near-UV light. The protocol required that cells be labeled prior t o exposure to acri- dine plus near-UV light, since it is known that this treat- ment renders the outer membrane permeable to the enzyme lysozyme (Wagner et a/., 1980) and thus poss- ibly to lactoperoxidase as well. After labeling, cells were exposed for varying lengths of time to near-UV light in the presence of 25 pg/rn/ acridine. A sample receiving n o light was maintained as a control. The labeled proteins were then analyzed by SDS poly- acrylamide gel electrophoresis to detect any modifica- tions in the electrophoretic mobility of the labeled species or the loss of any of the proteins.

Figure la shows a typical gel profile obtained for E. coli AB1157 exposed to near-UV light for 0, 3, 9 and 12 min. Cells exposed for 3 min to near-UV light, cor- responding to greater than 95% survival of c.f.u.. are not detectably altered in the outer membrane gel pro- file from the unexposed control cells. However, samples exposed to near-UV light for 9 and 12 min, corresponding to less than 30% survival of c.f.u., show

a differential loss of several protein bands, as indi- cated by the letter L to the left of the control sample. Another interesting effect observed in Fig. la is the appearance of a new band (marked N) in the low mol wt region for the samples exposed for 9 and 12 min to acridine plus near-UV light.

Figure 1. Sodium dodecylsulfate polyacrylamide gel pro- files of (a) E . coli AB1157 cells and (b) E . coli F cells labeled with '*'I in the presence of lactoperoxidase. The numbers above each column indicate the minutes of exposure to near-UV light in the presence of 25 pg/m/ acridine. Bands that are reduced in intensity by treatment are indicated by L; those that appear as new bands upon treatment are indicated by N. Proteins used as mol wt standards (column M) are as follows: a, 200000; b, 150000; c, 92000;

d, 69000; e, 53000; f, 46000; and g, 23000 daltons.

Research Note 257

The results of a similar experiment conducted with E. coli F are shown in Fig. Ib. Again, several protein bands are missing (marked L) and in this case more than one new band (marked N) is seen in the low mol wt region for the treated samples. These data suggest that acridine plus near-UV light is selective in its action on the '251-labeled outer membrane proteins of E . coli.

Bacteriophage adsorption studies

The outer membrane of E . coli contains proteins essential for the active or facilitated transport of various metabolites. Certain outer membrane proteins also function as receptors for the attachment of coli- cins and bacteriophage. The tonA gene codes for an 85000 dalton outer membrane protein that is involved in the binding and transport of iron-ferri- chrome, the sensitivity of cells to colicin M, and the adsorption of phage T1 and phage 480 (Braun et a/., 1973; Hantke and Braun, 1975; Hancock and Braun, 1976). This protein also serves as the receptor for phage T5, which was utilized in the present studies.

The adsorption kinetics of phage T5 to E. coli F were used to monitor the activity of functional recep- tor protein following treatment with acridine plus near-UV light. Cells were exposed for 12 min in the presence of 25 pg/m/ acridine. An unexposed sample was maintained as a control. The treated and control cultures were then mixed with phage T5 at a multi- plicity of infection (m.0.i.) of approximately 20 p.f.u. per cell and incubated at 37°C. At various times a por- tion of the sample was centrifuged to remove cells and adsorbed bacteriophage and the supernatant was then titered for unadsorbed p.f.u. using untreated E. coli F as plating cells. The results of one such experiment, presented as percent unadsorbed bacteriophage vs. time of adsorption, are presented in Fig. 2a. For early incubation times there is very little difference in the adsorption kinetics for phage T5 to control and treated cells. For longer times, however, there is a substantially greater fraction of unadsorbed bacterio- phage in the supernatant of the culture of cells that were treated with acridine plus near-UV light. The difference between treated and control cells is ap- proximately a factor of 10 for adsorption times longer than 10min. These data indicate that the functional integrity of the phage T5 receptor on the surface of E. co/i F cells is damaged by treatment with acridine plus near-UV light.

A strikingly different result was obtained for the adsorption of phage BF23 to E . coli AB1157 cells. The receptor for phage BF23 is a 60000-dalton outer membrane protein which also serves for BI2 binding and for the attachment of colicin E (Buxton, 1971; Jasper et a/., 1972; Sabet and Schnaitman, 1973). Figure 2b shows the adsorption kinetics for phage BF23 to untreated E. coli AB1157 cells and to cells treated with acridine plus near-UV light. The con- ditions for treatment and for adsorption were the same as for Fig. 2a. In this case, there is no discern-

( a ) Phage T5 Control cells

0 Treated cells

0 5 10 15 20

Minutes of adsorption

Minutes of adsorption

Figure 2. Adsorption of (a) phage T5 to E . coli F cells and (b) phage BF23 to E . coli AB1157 cells. (0) Adsorption kinetics of bacteriophage to control cells; (0) adsorption kinetics to cells previously treated for 12 min with near-UV light in the presence of 25pg/mt acridine. Data are graphed as the percent unadsorbed bacteriophage as a function of time after the bacteriophage were mixed with

the host cells.

ible difference between adsorption to control and treated cells, showing that the functional integrity of the phage BF23 receptor is not modified by exposure to acridine plus near-UV light. Thus, bacteriophage adsorption studies also establish a degree of selec- tivity in the action of acridine plus near-UV light on the outer membrane components of E. coli.

DISCUSSION

The analysis of '"I surface-labeled E. coli cells by SDS polyacrylamide gel electrophoresis revealed that

258 STEPHEN WAGNER and WALLACE SNIPES

certain outer membrane proteins are lost or modified in some manner upon treatment with acridine plus near-UV light, whereas other protein species appear to remain unaltered. There are several mechanisms whereby damage induced by acridine plus near-UV light could show up as a missing band on the gel profile. One possibility is that the protein becomes crosslinked either to another identical protein mol- ecule or to some other protein, thereby changing its electrophoretic mobility. If the newly formed, cross- linked molecules are heterogeneous in structure, they could migrate at various rates and go undetected on the polyacrylamide gel. There is existing evidence that the photodynamic action of fluorescein isothiocyanate plus visible light does in fact crosslink proteins in red blood cell (Lepock e t al., 1978) and herpes simplex virus (DeLuca et al., 1981) membranes.

Another possibility is that acridine plus near-UV light causes degradation of membrane proteins, yield- ing smaller polypeptides that migrate more rapidly. Again, these would not appear as distinct bands on the gels if their sizes were heterogeneous or if degra- dation was extensive. The light smear of radioactivity observed for treated samples is consistent with such an effect. On the other hand. if a protein species were cleaved at a small number of distinct sites, the resul- tant polypeptides would migrate as distinct bands of lower mol wt. This is a possible explanation for the origin of the new bands (marked N) on the gels of Fig. 1.

I t is also possible that outer membrane proteins may be released from the membrane by acridine plus near-UV light treatment and lost entirely when the cells are concentrated by centrifugation. The release of a protein could result from some modification of its interaction with phospholipids or with other proteins in the outer membrane. This could be due to a direct damage to the protein, changing its conformation or charge properties, or to photochemical damage to boundary lipids, thereby lessening the interactions that hold the protein in place. It also seems likely that peptide cleavage at a single site, particularly for a transmembrane protein, could result in release of the components from the membrane.

The adsorption of phage T5 to untreated E. coli AB1157 cells follows approximately pseudo first order kinetics up to about 5 min, after which a decrease in the apparent adsorption rate is observed. This curva- ture is probably due to reversibility in the adsorption process, since it was observed even for m.0.i. values as low as 0.1 p.f.u./cell (data not shown) and therefore cannot be due to saturation of receptor sites on the cell surface. The initial adsorption rate of phage T5 to cells treated with acridine plus near-UV light is only slightly different than to control cells, but at later times the apparent rate of adsorption to treated cells decreases more rapidly than to control cells. One possibility for this effect is that a significant fraction

of the receptor proteins is totally nonfunctional or missing, so that saturation of available receptor sites occurs earlier for adsorption to the treated cells. Another possibility is that most or all of the receptor molecules are damaged in such a way that the stab- ility constant for interaction of the bacteriophage with the receptor is reduced. For a reversible binding pro- cess, this would also appear as an increase in the fraction of unadsorbed bacteriophage at longer adsorption times.

Both the electrophoresis studies and the bacterio- phage adsorption studies presented here reveal that some outer membrane proteins are damaged by treat- ment with acridine plus near-UV light while others appear unmodified. This specificity could be due to an inherent difference in reactivity of the proteins with the active species produced during treatment, to dif- ferent susceptibilities of the proteins to release from the membrane when boundary lipids are damaged, or to some other factor(s) unknown at the present time.

Acknowledgements-We acknowledge Dr. James McCor- quodale for helpful discussions on some aspects of this project. This work was supported by the National Insti- tutes of Health and the Department of Energy.

REFERENCES

Arntzen, C. J., P. A. Armond, C. S. Zettinger, C. Vernotte and J. M. Briantais (1974) Biochim. Biophys. Acta

Bayse, G. S. , A. W. Michaels and M. Morrison (1972) Biochim. Biophys. Acta 284, 30-33.

Braun, V., K. Schaller and H. Wolf (1973) Biochim. Bio- phys. Acta 323, 81-97.

Buxton, R. S . (1971) Mol. Gen. Genet. 113, 154156. DeLuca, N., D. Bzik, S. Person and W. Snipes (1981)

Hancock, R. E. W . and V. Braun (1976) J . Bacteriol.

Hantke, K. and V. Braun (1975) F E B S Lett . 49,

Haustein, D. (1975) J. Immunol. Meth . 7, 25-38. Hogg, N. M. (1974) Proc. Nat l . Acad. Sci. U S A 71,

Jasper, P. E., E. Whitney and S. Silver (1972) Gener.

Laemmli, U. K. (1970) Nature (London) 227, 68&685. Laskey, R. A. and A. D. Mills (1975) Eur. J . Biochem.

Lepock, J. R., J. E. Thompson, J. Kruuv and D. F. H. Wallach (1978) Biochem. Biophys. Res. Commun. 85, 344350.

Person, S . and R. C . Bockrath (1964) Biophvs. J. 4,

Sabet, S. F. and C. A. Schnaitman (1973) J . Bid . Chem.

Wagner, S., A. Feldman and W . Snipes (1982) Photo- chem. Photobiol. 35, 73-81.

Wagner, S., W . D. Taylor, A. Keith and W. Sni- pes (1980) Photochem. Photobiol. 32, 771-779.

Wanda, P., J. Cupp, W. Snipes, A. Keith, T. Rucinsky, L. Polish and J. Sands (1976) Antimicrob. Agents Che- mother. 10, 96101.

347, 329-339.

Proc. Natl . Acad. Sci. U S A 78, 912-916.

125,409-41s.

301-305.

489492.

Res. 19, 305-312.

56, 335-341.

355-365.

248, 1797-1806.