Membrane Mutation Associated with

Transcription

1 JOURNAL OF BACrEDOLOGY, Apr. 1972, p Copyright 1972 American Society for Microbiology Vol. 11, No. 1 Printed in U.SA. Membrane Mutation Associated with Sensitivity to Acriflavine in Escherichia coli HAKOBU NAKAMURA AND ATSUSHI SUGANUMA Biological Institute, Faculty of Science, Konan University, Kobe, Japan, and Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto, Japan Received for publication 28 December 1971 The role of plasma membrane on the acriflavine sensitivity of Escherichia coli was studied. '4C-uracil incorporation into ribonucleic acid fraction by spheroplasts was more sensitive to acriflavine in the acriflavine-sensitive strain (genotype acra) than in the acriflavine-resistant (genotype acra+) strain. There was no difference between two types of cells in the response to osmotic shock, phage sensitivity, and other treatments used to investigate the structure and stability of cell wall. Differences in the electron-microscopic figures between acra and acra+ cells was found in the plasma membrane, surface area just below the membrane, and ribosomal aggregation, when cells were treated with acriflavine. It is concluded that a primary site of acriflavine action is on the plasma membrane, and the acra mutation is mediated by it. On the basis of the present results, it is evident that differences in the acriflavine binding and the sensitivity to phenethyl alcohol and sodium dodecyl sulfate between the acra and acra + strains, previously reported, are attributable to a structural difference in the plasma membrane between the two strains. Since the discovery that under certain conditions the sex factors of Escherichia coli male (F+) cells are effectively eliminated by the treatment with acridines (9), the action mechanism of the dyes on episomes has attracted much attention. It is reported that the stability of episomes depends upon the response on the host cell to acridines (28). Previously, one (H.N.) of us found that the cellular response to acriflavine (AF), a derivative of acridine, of E. coli K-12 was determined by a gene acra located near the lac gene (17, 18). The acra mutants accumulate more and thus are more sensitive to acriflavine and other basic dyes than acra+ strains (15, 16). The AF-sensitive mutants are also sensitive to lipophilic substances such as phenethyl alcohol and sodium dodecyl sulfate (18, 19). A possible inference from this evidence is that the structure determined by acra resides in the surface membrane of cells. The purpose of the present study is to determine which is more responsible for the AF sensitivity of cells, plasma membrane or cell wall. The results show that the acra mutation affects the plasma membrane. MATERIALS AND METHODS Bacterial strains. Strain N43 is an acra (AFsensitive) mutant of E. coli K-12 (17). Strain N9 is 329 an acra + (AF-resistant) recombinant from a cross W1895 (HfrC acr+, AF-r) x N43 (F- acra), interrupted at 4 min after onset of the mating. Therefore, the chromosome of N9 is largely in common with that of N43 (17). Strain N562 is an acra+ revertant of N43 (18). Phage experiments. Phages Ti, T2, T4, T5, and T6 were propagated in W1895, and their titer was determined with this bacteria. Mutants of N43 resistant to phage Ti or T4 were isolated from the phage-lysed zones of the phage-infected bacteria on plates and purified. Determination of efficiency of plating and rate of adsorption of phages to bacteria were carried out by the methods of Adams (1). Media. PGY broth contained polypeptone (1 g; Daigo-eiyo), yeast extract (3 g; Daigo-eiyo), NaCl, (3 g), and glucose (1 g per liter of water), and was adjusted to ph 7.4 with 1 N NaOH. Synthetic medium 3XD, used for the spheroplast preparation, was essentially identical to that of Fraser and Jerrel (8), consisting of Na2HPO4(1.5 g), KH2PO4 (4.5 g), NH4Cl (1 g), 1% gelatin (3 ml), Casamino acids (15 g), glycerol (3 g), 1 M MgSO4 (1 ml), and.1 M CaCl2 (1 ml per liter of water). The minimal medium SI-glucose has been described previously (17). The media were solidified with 1.5% powdered agar. AF stock solution was prepared by dissolving crystallized AF (Tokyo Kasei Co.) in sterilized water; to minimize decomposition, the stock solution was not steamed. Spheroplast preparation. Cells exponentially growing in 3XD medium were washed and suspended (ca. 5 x 18 cells/ml) in fresh 3XD supple- Downloaded from on January 27, 219 by guest

2 33 NAKAMURA AND SUGANUMA J. BACTERIOL. mented with.3 M sucrose, and lysozyme and ethylenediamine-tetraacetate (EDTA) were added to make 1 Ag/ml each. By microscopic observation, it was confirmed that all the cells became spheroplasts during 1 min of treatment at 2 C. Spheroplasts were washed with the sucrose-supplemented 3XD before experimental use, and the concentration was turbidimetrically adjusted to a standard. Radioisotopic experiments. Intact cells and spheroplasts were inoculated into prewarmed S,- glucose with or without.3 M sucrose, and 1 MCi of '4C-uracil per ml was added at zero time. Samples (.2 ml) were withdrawn at intervals and added to 3 ml of cold 5% trichloroacetic acid solution. After 3 min at C, cells and spheroplasts were collected on membrane (.45-,um pore size) filters, washed three times with the cold trichloroacetic acid solution, and dried. The radioactivity incorporated was assayed by a liquid scintillation counter. Electron microscopy. Cells were sampled from 1-min PGY cultures, some containing 1,g of AF per ml. After centrifugation, the pellet was sandwiched with soft agar and fixed with cold s-collidine (5) osmiumtetroxide, followed by treatment with uranylacetate by the method of Kellenberger (11). After dehydration, the specimens were embedded in Epon epoxy resin (12) and sectioned by using Porter- Blum microtome (MT-1). The electron microscope used was a Hitachi model 11-A. RESULTS AF sensitivity of the spheroplasts. To examine whether the cell wall was essential for the AF resistance, 4C-uracil incorporation into ribonucleic acid (RNA) was measured at 37 C, because the RNA synthesis in vitro has been shown to be more sensitive to proflavine, a derivative of acridine, than deoxyribonucleic acid (DNA) synthesis (1). Figure 1 indicates that the uracil incorporation was less sensitive to AF in strains N9 and N562 than in N43, both with intact cells and with spheroplasts. Hence, it is inferred that the acra + -controlled resistance mechanism does not reside in the wall. But, since it is well known that in gram-negative bacteria a part of cell wall may remain adhering to protoplast, even after the treatment with lysozyme- EDTA (13, cf. 3; we also ascertained this with the present samples) we sought cell wall changes in the AF-sensitive cells. The following treatments are known to be useful for testing the stability of the cell wall: exposure of cells to (i) osmotic shock (21). (ii) lysozyme (24), (iii) T phages (21, 3), (iv) EDTA (21, 22, 32), (v) penicillin (21), and (vi) colony pigmentation caused by the triphenyltetrazorium chloride reduction (2, 21). The cells of strains N43, N9, and N562 in exponential and stationary-growth phases were subjected to each of these treatments. Sensitivity of the cells to the agents was estimated by counting viable cells in samples withdrawn at intervals. Phage adsorption to bacteria and efficiency of plating were examined with phages Ti, T2, T4, T5, and T6. Effect of lysozyme was tested in the presence of EDTA or with the cells frozen in a dry ice-acetone mixture and then thawed. By any of these treatments, no significant differences could be noted between the acra and acra+ strains. In addition, mutants of N43 selected for resistance to phage Ti or T4 and penicillin were as sensitive to AF as the parent strain. Therefore, it is concluded that the AF resistance of acra+ spheroplasts is not due to the wall substances which may partly remain on the protoplasts, and thus the resistance mechanism must be situated in the plasma membrane. In Fig. 1, low concentration of AF stimulates the "4C-uracil incorporation into RNA, suggesting a special action of small amount of AF to the regulatory mechanism of RNA synthesis. Electron microscopy experiments. Since the acra mutation seems to control the nature of plasma membrane, comparison was made for its electron-microscopic structure. Both N43 and N9 showed the normal unit membrane structure, and no essential difference was noticed between the two strains (Fig. 2 and 6). N562 (picture not shown) also showed the same structure. However, when cells treated with AF were observed, the cytoplasmic surface region of N43 became low in electron density, and a granular aggregate of ribosomes was formed (Fig. 3). Furthermore, a lamellar structure was induced by AF in the plasma membrane of N43 (Fig. 4 and 5). In N9 (Fig. 7) and N562 (Fig. 8), on the other hand, no such lamellation was induced by AF. It should be stressed that AF did not cause any electron-microscopically observable effect on the wall and interior of cytoplasm (including nuclear region). DISCUSSION The intact cells of acra mutants are more sensitive to AF than are the intact cells of the wild type (acra+). The present results indicate that the difference in the AF sensitivity between the two strains can be attributed to a difference in the nature of plasma membrane between them. This is quite different from sensitivity of E. coli to novobiocin, actinomycin, and other antibiotics, which is determined by a chemical nature of the cell wall, particularly lipopolysaccharide layer (24, 29). Downloaded from on January 27, 219 by guest

3 VOL. 1 1, 1972 ACRIFLAVINE SENSITIVITY IN E. COLI *X6 E- 4 O 3 I a X2 E Q l U )2 i5 '1 i5,1.,2 Downloaded from Time in FIG. 1. Effect of AF on the '4C-uracil incorporation into the acid-insoluble fraction of intact cells and spheroplasts of strains N43, N9, and N562. (A, B, and C) Intact cells of N43, N9, and N562, respectively; (D, E, and F) spheroplasts of N43, N9, and N562, respectively. AF concentration (,ug/ml) indicated on each curve. (*) counts per minute of acid-insoluble fraction from cell or spheroplast suspension standardized for optical density at 575 nm. The acra cells bind more AF than do the acra+ cells (15, 16). AF uptake by the cells could be restricted by at least four parameters: binding in cell wall, permeation through a barrier in the wall, binding on plasma membrane, and permeation through a barrier in the membrane. The results of the experiments in this paper reveal that the difference in AF-binding capacity between the acra and acra+ cells is determined by a difference in the characters of the plasma membrane, but not the cell wall, between the two classes of cells. Silver et al. (26) found that the order of relative binding, but not permeability, efficiencies of acridines is the same as that for relative effectiveness in most inhibitory effects. This suggests that the AF absorption controlled by gene acra is associated with the binding sites on the plasma min membrane, and the biological effects of AF controlled by gene acra are determined by an interaction between AF molecules and sites (receptors). Phenethyl alcohol is more toxic for growth of gram-negative bacteria than that of gram-positive bacteria, and it has been suggested that the sensitivity of gram-negative bacteria is due to a destruction of the lipid layers in the wall by the alcohol (6). Treatment with phenethyl alcohol of cells also results in an increased uptake of AF in acridine-sensitive and resistant strains (26, 27). On the other hand, a rapid, increased uptake of AF takes place during the first stage (adsorption) in T-even phage infection of E. coli cells (25). The phage adsorption on the receptors residing in the cell wall is considered to cause a change in the wall on January 27, 219 by guest

4 2 M 2 p. *~~~~~~~~~~~~~~~~~~~~~.. -,m W. Ab~ ~~,W ~ "I.IN ~~~~~~~~~ 9' Downloaded from K4 3.2 p on January 27, 219 by guest FIG. 2. Section of strain N43 without AF treatment. (W) Cell wall, (M) plasma membrane, (N) nucleus. x6,ooo. FIG. 3. Section of strain N43 after AF treatment. (W) Cell wall, (M) thick plasma membrane, (N) nucleus. x6,. FIG. 4. Part of Fig. 3 (1) enlarged. (M) Thick plasma membrane of lamellar structure. xl1,. FIG. 5. Section of the same specimen as Fig. 3. (M) Plasma membrane of lamellar structure, (G) aggregate of cytoplasm. x1,. 332

5 VOL. 11, 1972 ACRIFLAVINE SENSITIVITY IN E. COLI /p P. j, I t, 'k-1n1 ae, SftJ..'.,li., v -l M- 4 4,,t t,.., '4 Downloaded from on January 27, 219 by guest 8 $ v J.,. A I FIG. 6. Section of N9 without AF treatment. (W) Cell wall, (A) plasma membrane, (N) nucleus. x6,o. FIG. 7. Section of N9 after AF treatment. (W) Cell wall, (M) plasma membrane, (N) nucleus. x6,. FIG. 8. Section of N562 after AF treatment. (W) Cell wall, (Al) plasma membrane, (N) nucleus. x6,.

6 334 NAKAMURA AND SUGANUMA J. BACTERIOL. structure (2). As Brown (7) also has pointed out, the bacterial cell wall must function as an ion exchanger (AF being cationic) and provide a general nonspecific restriction to diffusion. Therefore, a structural change of the cell wall may be one of the parameters to increase the AF uptake by phenethyl alcohol and phages. The actinomycin D sensitivity studied by Sekiguchi and lida (24) is a striking example for this problem. The actinomycin-sensitive mutants isolated by them take up much more actinomycin than the actinomycin-resistant (wild type) strains. However, the difference in actinomycin absorption comes from a structural difference in the cell wall (probably lipopolysaccharide layer; 24, cf. 29). Electron microscopy observation in this paper shows that a striking difference of acra cells from acra+ cells, when treated with AF, is the lamellation of plasma membrane. However, AF also causes a loss of electron-dense materials just below the membrane surface and more granular aggregate forms of ribosomes in the acra cells (Fig. 4 and 5). The previous experiment of Nakamura (15) demonstrated that AF uptake by the acra and acra+ cells is not dependent on the active transport system but is dependent on binding. Therefore, it is probable that AF binds primarily on the plasma membrane and there causes specific biological effects, but the mutant membrane allows more AF to diffuse to cytoplasm, since a larger amount of AF binds outside the mutant membrane than outside the wild-type membrane. (In this case, it is unknown whether the permeability to AF is higher in acra membrane than in acra+ membrane.) Gene acra determines sensitivity not only to acridine but also to phenethyl alcohol and sodium dodecyl sulfate (17, 18). On the basis of the present results, the sensitivity to these lipophylic substances determined by the allelism of acra gene is also attributed to a nature of the plasma membrane, although the chemicals destroy the lipid layers of the wall, as stated above (4, 6). Colicin-tolerant (tot) mutants are common to acra mutants in regard to high sensitivity to AF and detergents (14-19), and large uptake of AF as compared to the wild-type strains (14, 16, 27), although they differ from each other in chromosomal loci of the genes and other physiological characters (14-2, 31). Recently, Rolfe and Onodera (23) have found a missing membrane protein by gel electrophoresis with the tolc mutant. Nakamura (15) has proposed that gene acra+ determines synthesis of a basic protein which competitively interferes with the binding of AF, and its loss by mutation results in an increase in AF binding. It might be stated that the basic protein is a component of the plasma membrane. We are trying to isolate the plasma membranes from the acra and acra+ cells and to characterize the proteinous composition of them at present. ACKNOWLEDGMENT We are greatly indebted to Joji Ashida, Ehime University, for his interest and many helpful discussions. LITERATURE CITED 1. Adams, M. H Bacteriophages. Interscience Publishers, Inc., New York. 2. Anderson, T. F Bacterial viruses-structure and function, p In I. C. Gunsalus and R. Y. Stainier (ed.), The bacteria. Academic Press Inc., New York. 3. Bayer, M. E Areas of adhesion between wall and membrane of Ecsherichia coli. J. Gen. Microbiol. 53: Bayer, M., and T. F. Anderson The surface structure of Escherichia coli. Proc. Nat. Acad. Sci. U.S.A. 54: Bennet, H. S., and J. H. Luft s-collidine as a basis for buffering fixatives. J. Biophys. Biochem. Cytol. 6: Berrah, G., and W. A. Konetzka Selective and reversible inhibition of the synthesis of bacterial deoxyribonucleic acid by phenethyl alcohol. J. Bacteriol. 83: Bown, A. 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