JOURNAL OF BACTERIOLOGY, May 1973, p. 814-818 Copyright 0 1973 American Society for Microbiology Vol. 114, No. 2 Printed in U.S.A. Large Surface Blebs on Escherichia coli Heated to Inactivating Temperatures PAUL SCHEIE AND SUSAN EHRENSPECK Department of Biophysics, The Pennsylvania State University, University Park, Pennsylvania 16802 Received for publication 29 January 1973 Large surface blebs were observed with phase-contrast optics on Escherichia coli B/r and B,.1 heated to temperatures at which colony-forming ability was lost. Characterization of such blebs was consistent with the view that they were formed by a physical process and were bounded by the outer membrane of the cell. A hypothesis for thermal inactivation of E. coli is presented that places membrane damage near the primary lethal event. The primary lethal event in the thermal inactivation of bacteria remains to be determined. Enzyme-produced breaks in the deoxyribonucleic acid (DNA) have been implicated (15, 17) as have degradation of ribosomes (14), denaturation of proteins (12), and an altered membrane (6, 13). A role for the cell envelope has received recent support from this laboratory, where we have shown that Escherichia coli cells heated to 55 C become susceptible to lysozyme attack. Electron micrographs of E. coli heated to 70 to 100 C also have indicated damage to the cell envelope and, in particular, damage that resulted in occasional large blebs in the outer membrane (5). We report here what appear to be similar blebs prevalent on E. coli heated to temperatures in excess of 50 C, the temperature near which colony-forming ability begins to be lost. These blebs were observed both on a relatively heat-sensitive strain, B8,, and on a more heatresistant strain, B/r (2). Blebs were visible with phase optics and could be observed without fixation, staining, or embedding. Such observations have permitted additional characterization of the blebs which, in turn, suggest some properties for the outer portion of the cell envelope as well as a hypothesis for thermal inactivation of these bacteria. MATERIALS AND METHODS E. coli B/r and B,1 from local laboratory stock were grown in 10 ml of air-bubbled nutrient broth (Difco) at 37 C. During exponential growth, at concentrations of 107 to 108 cells/ml, 2-ml samples were removed and added to 8 ml of air-bubbled nutrient broth preheated and maintained in a large water bath at the specified 814 temperature. After various exposure times at the desired temperature, samples were placed on microscope slides at room temperature, covered with glass cover slips, and viewed in positive phase contrast with a Leitz microscope with a x90 high-contrast objective and x 10 oculars. Micrographs were made on Kodak Contrast Copy 35-mm film. Preheat treatment. Some cell cultures were treated with ethylenediaminetetraacetate (EDTA, final concentration 0.4%) and centrifuged at 8,000 x g for 3 min, and the pellet was suspended to the initial volume with fresh nutrient broth. Immediately, 2-ml samples were added to 8 ml of heated medium as before. Another preheat treatment consisted of washing log-phase cells by centrifuging them and suspending the pellet in double-distilled water to the initial volume. This was done twice, after which the suspended cells were warmed to 37 C before 2-ml samples were added to 8 ml of hot, double-distilled water. Postheat treatments. Some heated suspensions, cooled to room temperature (20 to 25 C), were subjected to one of several treatments. These included (with final concentration); sodium dodecyl sulfate, 0.4%; toluene, 10%; Formalin, 0.4%; Pronase, 50 yg/ml, ph 7 for 24 h; lipase, 50 gg/ml, ph 7 for 24 h; 7X, a cleaning solution with an anionic detergent (Linbro Chemical Co.), 1%; Brij 58 (Atlas Chemical Ind.), 1%; and EDTA, 0.4%. Also included in the postheat treatments were centrifugation at 8,000 x g for 5 min and storage at 4 C for several days. Survival of colony-forming ability. Samples from the heated suspension were diluted through nutrient broth blanks at 37 C, and 0.2-ml samples were plated in triplicate on nutrient agar plates. Colonies were counted after overnight incubation at 37 C. Squeezed cells. A drop of unheated cell suspension was placed on a microscope slide, and the cover slip was applied with as much thumb pressure as could be used without breaking the cover slip. The sample was then examined under a phase-contrast microscope.
VOL. 1149 1973 RESULTS LARGE SURFACE BLEBS ON HEATED E. COLI Characterization of blebs. Large blebs (Fig. la and b) appeared on the surface of E. coli B/r and B8.1 cells when these strains were heated for several minutes at temperatures above 50 C. According to Bridges et al. (2), B8.1 cells are more sensitive to heat than are B/r cells. Virtually all B/r cells, and about 90% of the B8.1 cells heated for 10 min at 55 C, had large blebs on their surfaces. Several aspects of the large blebs shown in Fig. la and b were typical of those found on these heated cells. (i) Blebs were spherical, and (ii) there were only one or two blebs on most cells. A bleb on two cells not completely separated almost invariably was located where the two cells were joined. Blebs on short cells (not shown) were occasionally located at the ends. (iii) The surface area of the blebs, determined by measuring their diameters and by assuming they were spheres, usually was 10 to 50% that of the cell, although the blebs made contact with only a limited portion of the cell surface. (iv) The concentration of material within the blebs was close to that of the medium vif 7Aaw *'Pt_ W-4 A:Pb,.. *i s Z g B~~~~~~.4, : r g. as indicated by the low contrast on the micrographs of Fig. la and b. At the same time, contrast in the cell remained high. This is consistent with the view that the boundaries of blebs were formed of material from the outer portion of the envelope, whereas the plasma membrane still confined most of the cytoplasm within the original rod. Other characteristics of large blebs were obtained by using slight variations in the treatment or by additional treatments to heated cells. Cells pretreated with EDTA still formed blebs when the EDTA was removed before the cells were heated. EDTA has been reported to remove up to 50% of the lipopolysaccharides in the outer membrane of E. coli (9). Therefore, if blebs were formed from the liposaccharide portion of the outer membrane, they were formed from the fraction remaining or the EDTA damage was quickly repaired. Blebs on heated cells could be destroyed by subsequent treatment at room temperature with the detergent sodium dodecyl sulfate, with EDTA, and with toluene. They were not destroyed by treatment with Formalin, Pronase, * get'' '-. 4~~~ t4t..4. 'tue IA! i' *" *g S? k 0 * S - - :ssar*, i Ut 4.* 4't..,_.. 815 FIG. 1. Phase micrographs of blebs on E. coli (scale marker = 2 pm). (a) E. coli B/r heated 10 min at 55 C; (b) E. coli B,-1 heated 10 min at 55 C; (c) E. coli Bir heated 5 min at 53 C after which BSA was added to a final concentration of 5%; (d) E. coli Bir squeezed between slide and coverslip at room temperature (20 to 25 C).
816 SCHEIE AND EHRENSPECK J. BACTERIOL lipase, centrifugation, 7X cleaning solution, detergent Brij 58, storage at 4 C for several days, or by boiling for 10 min. Bovine serum albumin (BSA) permeated some blebs and was excluded from others. When small amounts of BSA (5% final concentration) were added to suspensions of B/r cells at room temperature subsequent to having been heated for 3 min at 53 C, some of the smaller blebs appeared bright under positive phase contrast (Fig. lc). This indicates that the BSA raised the refractive index of the medium above that of the contents of these blebs; consequently, BSA must not have penetrated them. Large blebs were never found to be bright, not even in a medium containing 30% BSA, an amount sufficient to cause unheated cells to appear bright. The BSA must have permeated the large blebs so that the internal refractive index increased along with that of the medium. This also implies that the spherical shape of the blebs was maintained by the rigidity of the bleb wall rather than by a pressure differential. These observations raise the possibility that in the early stages of formation the bleb walls were impermeable to molecules the size of BSA, but that the permeability changed when the blebs became large. It is also possible, however, that the blebs that were impermeable to BSA were bounded by a different material that broke apart upon continued heating so that only the large, permeable blebs remained. Variation in bleb formation with temperature. The appearance of blebs on cells heated to temperatures known to inactivate these bacteria suggested that bleb formation might be closely related to thermal death. Figure 2 shows the fraction of cells observed to be free of blebs after being heated, together with survival of colony-forming ability of cells heated to similar temperatures. Although there was not a precise correlation at a given temperature between the fraction of cells without blebs and the fraction able to form colonies, both features underwent large changes in the same 4-degree temperature range. A 10-min exposure at 51 C was gentle to the B/r cells and few blebs were observed, whereas the same exposure at 55 C inactivated almost all of these cells and produced about as many with blebs as were to be observed. With B8l, the more sensitive strain, these effects were noted at 49 and 53 C, respectively. Several suggestions can be advanced that could account for the small mismatch of the rate of bleb formation with the rate of inactivation. (i) Some blebs undoubtedly were missed in the counting process if they were eclipsed by the 0 I I 2 U) m zli - o m0 4 at 0 C,IL z x I~~~~ x (C) (d).001 l l l l *0 5 10 0 5 10 TIME HEATED (MIN) FIG. 2. Appearance of blebs on heated E. coli Bir (a) and E. coli B,1 (b) along with survival of colonyforming ability for heated E. coli B/r (c) and E. coli Bs-1 (d). A, 49 C; 0, 51 C; x, 53 C; 0, 55 C. cell to which they were attached, if they had broken, or if they had pinched off. (ii) Growth of the blebs may have been due to a different process than that which initiated them, and this growth may have required additional time. (iii) Different postheat treatment of the plated cells might have enhanced their survival. The persistence of a significant fraction of heated B8 l cells without blebs likely was the result of an outer membrane different from that on B/r cells. It is not clear whether blebs had formed and then burst or pinched off, or whether none were ever formed on some cells. Bleb formation. There are two reasonable mechanisms that could account for bleb formation. One involves a ballooning of the outer membrane as it is stretched or unfolded in response to pressure from within. The other is unbalanced synthesis, such as that known to produce small blebs after various other treatments (4, 7, 8). Cells fixed with Formalin (0.4% final concen-
VOL. 114, 1973 LARGE SURFACE BLEBS ON HEATED E. COLI tration) immediately upon transfer to an ice bath after 10 min at 55 C still had blebs. Similar results were obtained when the Formalin was added while the cells were still at 55 C. Furthermore, blebs were observed on cells heated to above 90 C for only a minute or two, cooled rapidly, and fixed with Formalin. The conclusions to be drawn from these experiments are that the blebs were not formed by unbalanced synthesis during the heating, while on the microscope slide, or as a result of the cooling when removed from the high temperature. In another set of experiments B/r cells were centrifuged out of the nutrient broth, washed, and resuspended in distilled water at 37 C for various times before being added to water at 55 C. Most cells heated for 10 min after being resuspended in water for 1 min had blebs. Cells that spent 10 min in water before being heated had large blebs that were more difficult to discern because of lower contrast. Cells left in water at 37 C for 90 min and then heated as described above had very few large blebs, but numerous, barely resolvable small ones. The decrease in the number of observed large blebs on cells kept in water for extended times before heating could have resulted from other changes in the cells, such as the leakage of small molecules before the cells were exposed to the elevated temperature. These data also are consistent with a physical basis for bleb formation. A slightly different experiment was performed to test the possibility that internal pressure was involved in bleb formation. Unheated cells were squeezed between a microscope slide and cover slip, the intention being to increase the internal pressure mechanically and, perhaps, produce blebs such as those seen on heated cells. Many cells so treated were severely disrupted, others escaped damage and appeared normal, and some showed blebs quite similar to those seen on heated cells. An example is shown in Fig. ld. Mechanically increased internal pressure did mimic the effects of high temperatures, and the argument for a physical basis for bleb formation becomes more convincing. DISCUSSION We would like to offer a hypothesis for thermal disruption of E. coli. The initial event we propose to be a denaturation of a protein (or proteins) in the cell envelope that leaves the rigid peptidoglycan layer weakened at one or two points, and that is sufficient to prevent 817 colony formation under the conditions employed here. The next event is a rupture of the plasma membrane, caused by the internal osmotic pressure, at the point at which the weakened cell wall no longer can constrain it. Enough cytoplasm then leaves through this opening to bring the internal and external osmotic pressures into balance. The exuded material, as it enters the periplasmic space, forces the initiation of a bleb in the outer membrane by a pressure differential across it. At some size the bleb breaks or becomes leaky, and most of the cytoplasmic material it contains diffuses out into the medium. The remaining, leaky bleb maintains its shape without a pressure differential, perhaps as a result of a phase change induced in the lipopolysaccharide similar to that reported by Lopes and Innes (10). So long as heat is applied, the opening in the membrane provides a channel for degraded material to escape. When the cell is cooled, the opening presumably could heal due to the mobility of membrane components and the natural tendency of phospholipids to form bilayers. Strain differences observed in heat inactivation and reflected in differences in the number of blebs observed are then the result of differences in the cell envelopes. According to this view, the more sensitive cells had fewer blebs because the outer membrane broke and permitted a more rapid diffusion of the cytoplasm into the medium. One might expect to find some strains with other bleb characteristics. Cells of other species heated in water are reported to survive better than those heated in a nutrient medium (16). This is consistent with the above hypothesis, because cells placed in water are reported to lose most small metabolites (3) and would, therefore, undergo a decrease in internal osmotic pressure. This should result in a lower tendency to rupture when heated. Moreover, one can predict from this that blebs should be less prevalent on cells heated under hypertonic conditions. It would be quite interesting to know whether there is a specific region of the cell envelope that has a high sensitivity to thermal stress. In unseparated cells, such as shown in Fig. la and b, the blebs were observed at the junction, suggesting such a weakness near the division plane. The more general observation that blebs did not appear consistently in any one position on single cells is not very helpful in this respect, because one could not determine either the time elapsed after a division or the state of development of a new division plane for a given cell.
818 SCHEIE AND EHRENSPECK J. BACTERIOL. Information about characteristics of blebs on heated cells from a synchronized culture would be useful. An interesting property of the outer membrane is suggested by the small area of contact between bleb and cell and by the small number of blebs on heated cells. de Petris (5) has pointed out that the thickness of the bleb envelopes corresponds to that of the normal, outer membrane and that over most of the surface of a heated cell the outer membrane is smooth and close to the adjacent inner layers. From this he inferred that the outer membrane unfolds rather than stretches when forming a bleb. Consequently, if on normal cells the outer membrane is folded and not firmly attached to the underlying layers, one would expect pressure from the inside during heating (or squeezing) to promote a general detachment of this membrane, rather than the formation of only one or two blebs of the type observed. Hence, it appears that part of the outer membrane is relatively free to slide laterally over the cell surface while being constrained from disengaging from adjacent, inner layers at more than a few points. Perhaps the lipopolysaccharide is free to slide and the lipoprotein regions of the outer membrane hold it to the rest of the cell. However, it must be pointed out that electron micrographs produced from freeze-etched cells give the impression that the outer membrane of E. coli is smooth, not convoluted (1, 11). It is not clear whether this would be true for the strains used in this study. Formation of blebs as reported here and by de Petris would be considerably more difficult to understand if the normal outer membrane were smooth and also had the thickness of a plasma membrane. LITERATURE CITED 1. Bayer, M. E., and C. C. Remsen. 1970. Structure of Escherichia coli after freeze-etching. J. Bacteriol. 101:304-313. 2. Bridges, B. A., M. J. Ashwood-Smith, and R. J. Munson. 1969. Correlation of bacterial sensitivities to ionizing radiation and mild heating. J. Gen. Microbiol. 58:115-124. 3. Britten, R. J., and F. T. McClure. 1962. The amino acid pool in Escherichia coli. Bacteriol. Rev. 26:292-335. 4. de Petris, S. 1965. Ultrastructure of the cell wall of Escherichia coli. J. Ultrastruct. Res. 12:247-262. 5. de Petris, S. 1967. Ultrastructure of the cell wall of Escherichia coli and chemical nature of its constituent layers. J. Ultrastruct. Res. 19:45-83. 6. Iandolo, J. J., and Z. J. Ordal. 1966. Repair of thermal injury of Staphylococcus aureus. J. Bacteriol. 91:134-142. 7. Knox, K. W., M. Vesk, and E. Work. 1966. Relation between excreted lipopolysaccharide complexes and surface structures of a lysine-limited culture of Escherichia coli. J. Bacteriol. 92:1206-1217. 8. Koike, M., K. Ilda, and T. Matsuo. 1967. Electron microscopic studies on mode of action of polymyxin. J. Bacteriol. 97:448-452. 9. Leive, L. 1965. Release of lipopolysaccharide by E.D.T.A. treatment of E. coli. Biochem. Biophys. Res. Commun. 21:290-296. 10. Lopes, J., and W. E. Innes. 1970. Electron microscopic study of lipopolysaccharide from an avian strain of Escherichia coli. J. Bacteriol. 103:238-243. 11. Nanninga, N. 1970. Ultrastructure of the cell envelope of Escherichia coli B after freeze-etching. J. Bacteriol. 101:297-303. 12. Rosenberg, B., G. Kemeny, R. C. Switzer, and T. C. Hamilton. 1971. Quantitative evidence for protein denaturation as the cause of thermal death. Nature (London) 232:471-473. 13. Russell, A. D., and D. Harris. 1967. Some aspects of thermal injury in Escherichia coli. Appl. Microbiol. 15:407-410. 14. Russell, A. D., and D. Harris. 1968. Damage to Escherichia coli on exposure to moist heat. Appl. Microbiol. 16:1394-1399. 15. Sedgwick, S. G., and B. A. Bridges. 1972. Evidence for indirect production of DNA strand scissions during mild heating of Escherichia coli. J. Gen. Microbiol. 71:191-193. 16. Strange, R. E., and M. Schon. 1964. Effects of thermal stress on viability and ribonucleic acid of Aerobacter aerogenes in aqueous suspension. J. Gen. Microbiol. 34:99-114. 17. Woodcock, E., and G. W. Grigg. 1972. Repair of thermally induced DNA breakage in Escherichia coli. Nature N. Biol. 237:76-79.