ELECTRON MICROSCOPY OF VESICLES PRESENT IN BACTERIAL LYSATES OF ESCHERICHIA COLI. x n rf s. Director of the Department of Biology

Transcription

1 ELECTRON MICROSCOPY OF VESICLES PRESENT IN BACTERIAL LYSATES OF ESCHERICHIA COLI APPROVED: Major Professor a Minor Professor x n rf s Director of the Department of Biology Dean of the Graduate S c h o o l \

2 ELECTRON MICROSCOPY OF VESICLES PRESENT IN BACTERIAL LYSATES OP ESCHERICHIA COLI THESIS Presented to the Graduate Council of the North Texas State University in Partial Fulfillment of the Requirements For the Degree of MASTER OF ARTS By James Elwood Shaw, B. A. Denton, Texas August, 1966

3 TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS iv Chapter I. INTRODUCTION 1 II. MATERIAL AND METHODS 15 Bacteriophage-lysed Cultures Lysis By Lysozyme Lysis By Penicillin Microscopic Examination III. RESULTS 22 IV. DISCUSSION 42 V. SUMMARY 51 BIBLIOGRAPHY 52 in

4 LIST OP ILLUSTRATIONS Figure Page 1. Lysis of Escherichia coli ATCC Lysis of Escherichia coli ATCC Lysis of Escherichia coli ATCC by Lysozyme Vesicle from Escherichia coli ATCC Lysate Vesicle from Escherichia coli ATCC Lysate Vesicle from Escherichia coli ATCC Lysate Vesicle from Escherichia coli ATCC Lysate Vesicle from Escherichia coli ATCC Lysate Vesicle from Escherichia coli ATCC Lysate 40 IV

5 CHAPTER I INTRODUCTION Bacteriolysis is the dissolution or lysis of bacterial cells. Lysis is accompanied by the loss of such vital functions as metabolism, growth, and reproduction and results in immediate death of the organisms involved (29). If lysis occurs without the help of an exogenous agent, the process is called autolysis. Autolytic destruction of bacteria frequently occurs in aging cultures and is apparently due to lytic enzymes that are capable of destroying bacterial cells from within (5). If lysis is due to an exogenous agent, the process is called heterolysis (34). Heterolytic agents may be physical, chemical, mechanical, or biological in origin and usually affect the bacterial cell from without. The distinction between autolytic and heterolytic agents or autolysis and heterolsis is sometimes difficult, especially if both processes occur simultaneously in the same bacterial culture. Endogenous agents liberated during autolysis become

6 exogenous agents? the latter may be capable of destroying bacteria by heterolysis (22). Regardless of the process involved, susceptibility to lysis by bacteriolytic agents depends on the chemical composition of the bacterial cell wall since this component of the bacterial cell serves as the substrate for the lytic agent (29). Cell walls of Gram-positive and Gram-negative bacteria differ in chemical composition, complexity, and susceptibility to bacteriolytic agents. Although the cell walls of Gram-positive and Gram-negative bacteria consist of essentially the same basic components (lipids, polysaccharides, and murein), Gram-negative cell walls are more complex in composition (24, 27). Cell walls of Gram-positive bacteria have a high content of polysaccharides and murein, whereas cell walls of Gram-negative bacteria have a high content of lipid material (29). The high lipid content in cell walls of Gram-negative bacteria probably makes them refractory to the action of lysozyme (29). Gram-positive organisms are readily lysed by treatment with lysozyme, whereas Gram-negative organisms are resistant to the action of lysozyme unless intact cells are specially pretreated to render them susceptible (17).

7 According to Weidel et_ajl. (33) the cell wall of Escherichia coli may be chemically fractionated into three distinct components. Two outer layers consist of lipoprotein and lipopolysaccharide and provide a cover for the inner mucopolymer complex (murein) which is adjacent to the cytoplasmic membrane. Only the two outer layers of lipoprotein and lipopolysaccharide are responsible for bacteriophage attachment. The inner mucopolymer complex is responsible for the structural rigidity of the bacterial cell; its presence in the cell wall of Escherichia coli is necessary for the typical rod shape of this organism. The mucopolymer complex consists of five characteristic components: muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid (37) found only in bacterial cell walls. The mucopolymer complex has also been called murein, glycopeptide, mucopeptide, or murein sacculus by other investigators (28,29). If the mucopolymer complex of bacterial cell walls is destroyed or sufficiently damaged, the cells lyse due to internal osmotic pressures. Penicillin, lysozyme, and bacteriophages are well known bacteriolytic agents of biological origin and are capable of lysing susceptible bacteria by partial or total destruction of the mucopolymer complex.

8 The apparent mechanism of action of penicillin differs from that of lysozyme and bacteriophages by either inhibiting the synthesis of murein (26, 30) or by interfering with the linkage of murein to the lipoprotein component of the cell wall (25). Lysozyme action does not interfer with cell wall synthesis but breaks 3(1, 4)-glucosamine linkages in the N-acetylglucosamine polymer of murein (1). The lytic action by bacteriophages is also due, at least in part, to an enzyme that has been characterized as a lysozyme (16). Weidel et a^l. (33) have successfully isolated and purified the mucopolymer of Escherichia coli cell walls and have demonstrated its degradation by lysozyme and phage enzyme. According to Delbruck (8), bacteriophages may lyse host cells by two methods. "Lysis from without" occurs when the phage-bacterium ratio is high (200:1). Since lysis by this method causes immediate dissolution of host cells, there is no apparent increase in the infective phage titer. "Lysis from within" occurs when the phage-bacterium ratio is low (1:1) The infective phage titer increases following lysis by this method since host cells are not destroyed before new phage progeny are produced.

9 Bacteriolysis may be prevented if susceptible organisms are suspended in hypertonic media prior to the addition of bacteriolytic agents? however, morphological changes usually accompany such treatment in hypertonic media and the terms "protoplast", "spheroplast", and "L-forms" of bacteria are used to describe these changes. The term protoplast refers to the living material of a plant cell. Bacterial protoplasts, by definition, are completely devoid of cell wall material. They may occur spontaneously in bacterial cultures (29) or be induced in the laboratory by treating normal cells (usually Grampositive cells) with cell wall degrading agents. Cocci remain coccoid in shape after conversion to protoplasts, but rod-shaped bacteria assume spherical shapes after removal of their cell walls. Bacterial protoplasts are capable of growth but not reproduction; they do not revert to normal cells containing cell wall material (20) nor do they adsorb bacteriophages to which normal cells are sensitive. Lederberg and Clair (19) list two criteria for the absence of a cell wall: (1) "osmotic fragility" (2) "loss of rigidity resulting in spherical or amoeboid shape."

10 Fitz-James (10) has shown that protoplasts of Bacillus megaterium are able to grow ten to twenty times their actual size and initiate a cell division, but do not divide. Weibull (32) has reported that lysozyme-induced protoplasts of Bacillus megaterium do not adsorb bacteriophages to which normal cells are sensitive. Bacteriophage development in protoplasts of Bacillus megaterium may occur if infection precedes treatment with lysozyme (3). L-form is a term introduced by Klieneberger (14, 15) when she discovered naturally occuring "minute granules" in cultures of Streptobacillus moniliformis. Klieneberger 1 s L-forms were stable after isolation and did not resemble the bacillary forms of Streptobacillus moniliforirtis. Instead, they resembled pleuropneumonia organisms of cattle in their morphology, growth requirements, and colony type. Bacterial L-forms usually arise when Gram-positive or Gram-negative bacteria are subjected to various bacteriolytic agents or to unfavorable environmental conditions (31). Bacterial L-forms, unlike protoplasts, are highly pleomorphic and may consist of granular, vesicular, and branched structures, Stable L-forms and protoplasts are incapable of reverting to the original cell types (20). Unstable L-forms, like spheroplasts (9), are capable of reverting to normal cells.

11 Hofschneider (13) has applied the term spheroplast to osmotically sensitive forms of Escherichia coli since these spherical forms, unlike the protoplasts of Gram-positive bacteria, retain some of their cell wall material after treatment with cell wall degrading agents. Spheroplasts are capable of growth and division in hypertonic media and revert to normal cells in the absence of the inducing agent. Lederberg and Clair (19) have suggested that penicillininduced spheroplasts of Escherichia coli continue to enlarge in broth cultures but do not divide. Hirokawa (12) has shown that spheroplasts of Escherichia coli revert to rod-shaped cells and suggests that one spheroplast is capable of giving rise to more than one normal cell. Nermut and Svoboda (23) have shown that lysozyme-induced spheroplasts of Proteus vulgaris are capable of reverting to their normal rod shape. Carry, Spilman, and Baron (4) have shown that inactive bacteriophages are capable of inducing spheroplast formation in Escherichia coli in hypertonic sucrose media. Active phages lyse the spheroplasts in hypertonic media. Hofschneider (13) has reported the adsorption of bacteriophages to spheroplasts of Escherichia coli. The spheroplasts were induced by treating normal cells with

12 8 lysozyme, penicillin, phage enzyme or by depriving auxotrophs of diaminopiraelic acid. Although a considerable amount of work has been reported in the literature concerning the morphological changes of bacteria during the course of bacteriolysis, little has been mentioned about the products of these cells remaining after complete lysis. In many cases the light microscope is inadequate for such studies since the subcellular products remaining after complete bacteriolysis may be below its resolution. The advent of the electron microscope during the 1930"s made such studies possible since the limits of its resolution extend several orders of magnitude below that of the ordinary light microscope. High resolution of small objects followed further improvements in the electron microscope and new and improved methods of preparing specimens for observation. Shadow casting (35) or "negative staining" (2) with heavy metals has greatly enhanced the contrast of small objects making detailed observations possible. Hillier et sll. (11) and Wyckoff (38, 39) were among the first investigators to report electron-microscopic evidence of bacteriolysis. Hillier et al_. (11) have shown circular and elliptical structures in unstained preparations of phagelysed cultures of Escherichia coli.

13 9 Wyckoff (39) observed spherical granules in bacteriophage lysates of Escherichia coli and suggested they represented a stage in the development of bacteriophages,since phages were occasionally seen surrounding the granules. In 1958, seven years after Wyckoff's observations, Mercer (21) reported small vesicles in phage-lysed cultures of Escherichia coli and suggested they were formed by "the rounding-up of fragments of lysed bacterial membranes.,r "Since membranes of homogenized mammalian cells form similar vesicles. Mercer suggested this phenomenon indicated a general property of biological membrances. Differences between the products resulting from "lysis from without" and "lysis from within" have been reported by Cota-Robles (6) and Cota-Robles and Coffman (7). Following lysis from without with T2 phage, the cell wall and membrane of Escherichia coli are not extensively degraded and may be seen surrounding the cell after its intracellular contents have been lost. Occasionally small vesicles may be seen within the lysed cells, but are bounded only by single unit membranes. The products resulting from lysis from within are vesicles bounded by one unit membrane or vesicles consisting of concentric rings composed of triple-layered membranes.

14 10 The reports of Wyckoff (39), Mercer (21), Hillier (11), Cota-Robles (6), and Cota-Robles and Coffman (7) suggest an association between lysis by bacteriophage and the vesicles produced in phage-lysed cultures of Escherichia coli?however, only wyckoff (39) and Mercer (21) present electron-microscopic evidence of phage contact with these vesicles. Their electron micrographs fail to reveal the fine structure of these vesicles and their association with bacteriophages. It is the purpose of this thesis to report further studies on the vesicles appearing in phage lysates of Escherichia coli, phage attachment to these vesicles, and the presence of similar vesicles in lysozyme and penicillin lysed cultures.

15 CHAPTER BIBLIOGRAPHY 1. Berger, L. R. and Weiser, R. S., "The B-Glucosaminidase Activity of Egg-White Lysozyme," Biochimica et Biophysica Acta, XXVI (1957), Brenner, S. and Home, R., "Negative Staining Method for High-Resolution Electron Microscopy of Viruses," Biochimica et Biophysica Acta, XXXIV (1959), Brenner, S. and Stent, G. S., "Bacteriophage Growth in Protoplasts of Bacillus megatherium," Biochimica et Biophysica Acta, XVII (1955), Cary, W. F., Spilman, W., and Baron, L. S., "Protoplast Formation By Mass Absorption of Inactive Bacteriophage, Journal of Bacteriology, LXXIV (1957), Chatterjee, B. R. and Williams, R. P., "Cytological Changes in Aging Bacterial Cultures," Journal of Bacteriology, LXXXIV (1962), Cota-Robles, E. H., "Electron Microscopy of Lysis from Within of Escherichia coli by Coliphage T2," Journal of Ultrastructure Research, XI (1964), Cota-Robles, E. H. and Coffman, M. D., "Electron Microscopy of Lysis from Without of Escherichia coli," Journal of Ultrastructure Research, X (1964), Delbruck, M., "The Growth of Bacteriophage and Lysis of the Host," Journal of General Physiology, XXIII (1940), Diena, B. B., Wallace, R., and Greenburg, L., "The Production and Properties of Salmonella typhi Spheroplasts," Canadian Journal of Microbiology, X (1964),

16 Fitz-James, P. C., "Cytological and Chemical Studies of the Growth of Protoplasts," Journal of Biophysical and Biochemical Cytology, IV (1958), Hillier, J., Mudd, S., and Smith, A., "Internal Structure and Nuclei in Cells of Escherichia coli as shown by Improved Electron Microscopic Techniques," Journal of Bacteriology, LVII (1949), Hirokawa, H., "Biochemical and Cytological Observations During the Reversing Process from Spheroplast to Rod- Form Cells in Escherichia coli," Journal of Bacteriology, LXXXIV (1962), Hofscheider, P. EL, "Ti and Lambda Phage Adsorption on Protoplast-Like Bodies of Escherichia coli," Nature, CLXXXVI (1960), Klieneberger, E., "The Natural Occurrence of Pleuropneumonia- Like Organisms in Apparent Symbiosis with Streptobacillus moniliformis and other Bacteria," Journal of Pathogenic Bacteriology, XL (1935), Klieneberger-Nobel, E., "Filterable Forms of Bacteria," Bacteriological Reviews, XV (1951), Koch, G. and Dryer, W. J., "Characterization of an Enzyme of Phage T2 as a Lysozyme," Virology, VI (1958), Kohn, A., "Lysis of Frozen and Thawed Cells of Escherichia coli by Lysozyme, and their Conversion into Spheroplasts," Journal of Bacteriology, LXXIX (1960), Lederberg, J., "Bacterial Protoplasts Induced by Penicillin," Proceedings of the National Academy of Science, XLII (1956), Lederberg, J. and Clair, J., "Protoplasts and L-Type Growth of Escherichia coli," Journal of Bacteriology, LXXV (1958), Martin, H. H., "Bacterial Protoplasts-A Review," Journal of Theoretical Biology, V (1963), 1-34.

17 Mercer, E., "An Electron Microscopic Study on Thin Sections and Bacteriophage Grown on Agar Plates," Biochimica et Biophysica Acta, XXXIV (1959), Mitchell, P. D. and Moyle, J., "Autolytic Release and Osmotic Properties of Protoplasts from Staphylococcus aureus," Journal of General Microbiology, XVI (1963), Nermut, M. V. and Svoboda, A., "Reversion of Spheroplasts Produced by Lysozyme into Rods in Proteus vulgaris," Nature, CXCIII (1962), Perkins, H. R., "Chemical Structure and Biosynthesis of Bacterial Cell Walls," Bacteriological Reviews, XXVII (1963), Plapp, R. and Kandler, 0., "Zur Wirkungsweise Zellwandhemmender Antibiotica bei Gram-negative Bakteriem," Archiv fur Mikrobiologie, L (1965) Rogers, H. J. 'and Mandelstam, J., "Inhibition of Cell- Wall-Mucopeptide Formation in Escherichia coli by Benzlpenicillin and 6-[D(-)-alpha-aminophenylacetamido] penicillanic acid (ampicillin),,r Biochemical Journal, LXXXIV (1962), Salton, M. R. J., "Bacterial Cell Wall. IV. Composition of the Cell Walls of some Gram-Positive and Gram- Negative Bacteria," Biochimica et Biophysica Acta, X (1953), Stent, G. S., Molecular Biology of Bacterial Viruses, San Francisco, W. H. Freeman and Company, Stolp, H. and Starr, M. P., "Bacteriolysis," Annual Review of Microbiology, XV (1965), Strominger, J. L., Park, J. T., and Thompson, R. E., "Composition of the Wall of Staphylococcus aureus; Its Relation to the Mechanism of Action of Penicillin," Journal of Biological Chemistry, CCXXXIV (1959),

18 Thorsson, K. G. and Weibull, C.» "Studies on the Structure of Bacterial L-forms. Protoplasts, and Protoplast-like Bodies," Journal of Ultrastructure Research, I (1958), Weibull, C., "The Isolation of Protoplasts from Bacillus megatherium by Controlled Treatment with Lysozyme," Journal of Bacteriology, LXVT (1953), Weidel, W., Prank, H., and Martin, H. H., "The Rigid Layer of the Cell Wall of Escherichia coli Strain B," Journal of General Microbiology, XXII (1960), Welsh, M., "Lysis By Agents of Microbial Origin," Journal of General Microbiology, XVIII (1958), Williams, R. C. and Wyckoff, R., "Applications of Metallic Shadow-Casting to Microscopy,,r Journal of Applied Physics, XVII (1946), Work, E., "The Mucopeptides of Bacterial Cell Walls, A Review," Journal of General Microbiology, XXV (1961), Work, E. and Dewey, D. L., "The Distribution of Diaminopimelic Acid Among Various Microorganisms," Journal of General Microbiology, IX (1953), Wyckoff, R., "The Electron Microscopy of Developing Bacteriophage," Biochimica et Biophysica Acta, II (1948), Wyckoff, R., "Possible Immature Forms of Bacteriophage," Experentia, VIII (1950),

19 CHAPTER II MATERIALS AND METHODS Sterile technique was employed during the handling of all materials and organisms, except during the preparation of specimens for electron microscopy. Specimens were observed immediately after preparation or were refrigerated and observed at a later time on the same day. Growth media, glassware, and distilled water were autoclaved for 15 minutes at 12ic> c (250 F) for sterilization. Solutions of lysozyme, penicillin, EDTA, and tris buffer were sterilized by positive-pressure filtration through millipore filters of 0.22 micron porosity. Two bacteriophage-sensitive strains of Escherichia coli were used in this study, ATCC and ATCC Bacteriophage T4 was obtained from North Texas State University and was capable of lysing strain ATCC A bacteriophage capable of lysing strain ATCC was isolated from Denton, Texas, sewage. 15

20 16 Bacteriophage-lysec! Cultures Specimens of phage-lysed bacteria were removed from trypticase soy broth (TSB, Difco) or from plaques formed on trypticase soy agar (TSA, Difco) plates. Broth cultures were prepared by adding 0.1 ml of the organism (ATCC or ATCC 11775) to 250 ml of trypticase soy broth. After four to six hours of growth at 37 C, 0.1 ml of the specific bacteriophage suspension was added to the culture. The culture was incubated at 37 C for three weeks. Two controls were prepared; one contained ATCC and the other contained ATCC The controls were prepared according to the above procedure but were not inoculated with bacteriophage. Agar plates were prepared by pouring 20 ml of trypticase soy agar (15 gm agar/liter) into petri dishes. After the agar hardened, each plate was inoculated with 3.0 ml of liquid trypticase soy agar (7.0 gm agar/liter) at 45 C, which contained 0.1 ml of an 18 to 24 hour culture of the organism and 0.1 ml of the specific bacteriophage dilution. Ten (tenfold) serial dilutions of the bacteriophage suspension were made in distilled water. Two bacteriophage-free controls were prepared according to the same procedure. The plates were incubated at 37 C.

21 17 After 24 hours of incubation, the plates were removed from the incubator and examined- Petri plates with well isolated plaques and controls were covered with sterile, moist filter pads and incubated 48 hours at room temperature (26-30 C) Specimens from plague areas and from controls were removed during the incubation period at room temperature. Lysis By Lysozyme A lysozyme-tris buffer-edta system was used to lyse ATCC according to the procedure of Repaske (3). Thirty milliliters of a 12 to 18 hour culture (TSB) of the organism were centrifuged at 3000 times gravity (g) for 10 minutes. The supernatant was discarded and the pellet washed three times in 15.0 ml of distilled water. After each washing the cells were centrifuged for 10 minutes at 3000 times g and the supernatant material discarded. After the third washing, the pellet was resuspended in an aqueous solution containing 5.0 ml of EDTA {ethylenediaminetetraacetic acid, 0.8 mg/ml, ph 7.5) and 5.0 ml of tris buffer [2-amino-2-(hydroxymethyl)-1,3- propanediol, 100 im/ml, ph 8.0], After five minutes at room temperature, lysis was started by adding 5.0 ml of crystalline egg-white lysozyme (Nutritional Biochemicals Co.). The test tube containing the mixture was gently agitated for 15 minutes at room temperature. Aliquants were then removed for

22 18 electron-microscopic examination. Two controls were prepared according to the same procedure: one contained 10.0 ml of tris buffer and 5.0 ml of EDTA but did not contain lysozyme; the other contained 10.0 ml of tris buffer and 5.0 ml of lysozyme but did not contain EDTA. Lysis By Penicillin Penicillin "G" (Calbiochem) was used to lyse strain ATCC to the spheroplast stage according to the procedure of Lederberg (2). Complete lysis was effected by resuspendingthe spheroplasts in distilled water. A six hour culture (TSB) of the organism was washed two times according to the procedure under "Lysis By Lysozyme." After the second wash, the cells were resuspended in 250 ml of fresh media (TSB), which contained 20 per cent sucrose, 0.2 per cent magnesium sulfate, and 1000 units penicillin "G"/ml. The culture was incubated four to six hours (sufficient time for spheroplast formation) at 37 C. After spheroplast formation, 30.0 ml of the culture were removed and centrifuged at 3000 times g for 20 minutes. The supernatant was discarded and the pellet resuspended in 5.0 ml of distilled water to lyse the spheroplasts. Aliguots were then removed for electronmicroscopic examination. Controls were prepared according to the procedure above, but did not contain penicillin.

23 19 Microscopic Examination The cellular debris that accumulated in phage-lysed cultures during the three-week incubation period was removed by centrifugation. A ten milliliter aliquant of the culture was centrifuged at 8000 times g for twenty minutes. The pellet was discarded, and the material remaining in the supernatant was concentrated by centrifugation at 4-0,000 times g for one hour at 23 C. The small pellet formed by one hour of centrifugation was resuspended in 0.2 ml of the supernatant and was used for electron-microscopic examination. Bacteriophage-free controls were prepared according to the same procedure. 'Specimens from plaque areas and those from lysozyme and penicillin-lysed bacteria and their controls did not require purification prior to examination. All specimens for examination were collected on carboncovered stainless steel grids (200 mesh). Carbon was vaporized in a Mikros VE-10 vacuum evaporator and allowed to settle on collodion-covered grids. The collodion was subsequently dissolved by placing the grids in amyl acetate, leaving only the thin carbon film covering the grids. A small drop of the liquid material was placed on the surface of the grid (carbon side) and the excess removed by touching>the edge of the grid to a filter pad. Specimens

24 20 from plaques were collected by gently touching the carbon surface of the grid to the plaque area. All specimens were allowed to dry at room temperature prior to staining. The negative technique employed was similar to the procedure developed by Brenner and Home (1). Phosphotungstic acid was dissolved in glass distilled water to a concentration of 2.0 per cent. The ph of the solution was adjusted to 5.5 with 5N potassium hydroxide. Prior to use, one drop of the stain was placed in a depression slide and mixed with one drop of glass distilled water. The grids were inverted on the drop (specimen side in contact with the stain) and withdrawn after 30 seconds. Excess stain was removed by touching the edge of the grid to a filter pad? the film remaining on the grid was allowed to dry at room temperature. Electron micrographs were taken on an RCA EMU-3G electron microscope using a 50 p, objective aperture and operating at 50 kv.

25 CHAPTER BIBLIOGRAPHY 1. Brenner, S. and Home, R. w., "Negative Staining Method for High-Resolution Electron Microscopy of Viruses," Biochirnica et Biophysica Acta, XXXIV (1959), Lederberg, J., "Bacterial Protoplasts Induced by Penicillin," Proceedings of the National Academy of Science, XLII (1956), Repaske, R., "Lysis of Gram-Negative Bacteria By Lysozyme," Biochirnica et Biophysica Acta, XXII (1956),

26 CHAPTER III RESULTS Spherical vesicles were present in penicillin, lysozyme, and bacteriophage lysates of Escherichia coli. Vesicles were also present in bacteriophage-free broth cultures (controls) during the three-week period of incubation. Vesicles were not present in penicillin and lysozyme controls nor were they present in bacteriophage-free controls grown on solid media. After three days of incubation some of the cells were swollen or spherical in shape in control cultures grown on solid media. Usually no distinction could be made between the vesicles present in penicillin, lysozyme, or bacteriophage lysates. Only when the vesicles retained bacterial pili or flagella or when bacteriophages were in contact with vesicles could distinctions be made. Within a single lysate vesicles differed not only in aize but also in morphology. The diameter of most of the vesicles was 0.2 to 0.6 jj,;some were as large as 0.8 [a, and others as small as 0.1 (j,. While some of the larger vesicles contained smaller structures, others appeared to be empty. Evidence is 22

27 23 presented (Figures 3, 7) which might indicate that the smaller structures are expelled from larger ones. Some of the vesicles in phage-lysed cultures retained adsorbed bacteriophages (Figures 4,5, 6, 9). Occasionally bacteriophages were seen within vesicles (Figure 8). Whether the vesicles are cell wall and/or membrane in origin has not been determined. Vesicles were present in cells prior to complete disruption of the "cell wall" or during disruption of the bacterial cell. Some of the vesicles were limited by thick "cell wall" material, while others were surrounded by thin "membrane-like" structures. The flat or collapsed appearance of some of the vesicles might be due to their small size, the low contrast of the electron micrographs, or to the thickness of the preparation. Others may have been totally or partially collapsed due to the loss of cytoplasmic material. Some of the electron micrographs taken during the course of this work are presented on the following pages. The sequence in which the micrographs are presented does not indicate the stages that might have occurred during bacteriolysis. Figure 1 represents one cell of Escherichia coli ATCC during bacteriolysis by bacteriophage T4. The specimen was taken from a plaque area after 24 hours of growth at room temperature, and is magnified 198,000 times.

28 24 W- A Fig. 1 Lysis of Escherichia coli ATCC 11303

29 25 The bacterial cell, apparently still intact, represents the typical rod shape of Escherichia coli. Flagella are not present but bacterial pili may be seen extending from the periphery of the cell. Small "blebs" are also seen extending outward from the surface of the cell. One phage with its tail in a contracted state can be seen attached to the cell surface. Another phage may be seen within the cell. Its tail is extended rather than contracted. Since this micrograph does not represent a thin section of the bacterial cell, it must be realized that most of the material present is on the surface and not within the cell. Numerous vesicles of different sizes are visible on the surface of the cell. Some of the vesicles contain smaller structures. Since vesicles were not observed on the surface of untreated cells, the vesicles in this micrograph may represent extensive destruction or alterations of the cell wall and membrane. Figure 2 represents one cell of Escherichia coli ATCC during bacteriolysis. The specimen was removed from a plaque area after 48 hours of growth at room temperature. The bacteriophage responsible for plaque formation was"isolated from Denton, Texas,sewage. Although no bacteriophages are present around the surface of the cell, small structures resembling bacteriophage heads are present within the cell.

30 Fig. 2 Lysis of Escherichia coli ATCC

31 27 Large vesicles appear to be "budding" from the surface of the cell. Some of the vesicles have retained "cell wall" material which appears white in color and surrounds the vesicles. Two flagella are visible. One is in contact with the cell while the other appears to be detached. Autolytic destruction may have been responsible for the morphological alterations of this cell. Plaques are usually visible three to four hours after bacteriophage inoculation and represent a zone of lysis on the culture media. If the bacterial cell presented in this micrograph was resistant to bacteriophage attachment, it may have been affected by enzymes liberated from susceptible cells. The structures in this electron micrograph are 136,000 times actual size. Figure 3 represents one cell of Escherichia coli ATCC during lysis by lysozyme. Most of the specimens examined from lysozyme lysates did not resemble the structures revealed in this micrograph. The typical appearance of most vesicles in lysozyme lysates resembled those in autolytic cultures and in bacteriophage and penicillin lysates. This micrograph was included since it probably represents a stage during the bacteriolytic process. The typical rod shape has been lost and the cell has assumed a spherical shape prior to complete lysis. Extensive alterations in the surface

32 28 Li p.:.;. i-fi r Fig. 3 Lysis of Escherichia coli ATCC by Lysozyme

33 29 or interior of the cell are evident, but the limiting structures surrounding the entire vesicle are not completely damaged. The cell has probably collapsed due to the loss of its cytoplasmic constituents. Magnification is 150,000 times actual size. A vesicle from Escherichia coli ATCC and a bacteriophage are presented in Figure 4. This specimen was removed from a three week old broth culture and is magnified 370,000 times. It is difficult to determine if the bacteriophage is in contact with the surface of the vesicle since the resolution of this micrograph prevents identification of the phage tail fibers and core. Close observation reveals that the material limiting the vesicle is not as wide in the area around the phage tail as it is around the rest of the vesicle. This might indicate that cell wall material surrounds the vesicle and has been partially destroyed by phage enzyme in the area around the phage tail. It is of interest to note the alrac equidistant divisions in the material surrounding the vesicle and to compare the thickness of the material with that in Figure 5. If it were not for the electron dense material in the center,the vesicle would appear to be empty. This may be an

34 30 S&.&**SV>V*U t m. < iis : j^'a StSSf3Bimfr.?*i M^c". ^fo.^i t.'fev'; I S^wMra& I,-i^iIfcfffeWr f S ṛ. &&*' -.ih $t^r>?/?tv' : v-'.& &*;.. V^ifeeMji -mm ;.-& * mmm. MS" jv.ji * >, >. ;';«,:;Kir,:; *$& >i ' %Vv^- '- *- '? ' < :y : S'.% #AS#«BS3PI H -. - ^ v.:; Fig. 4 Vesicle from Escherichia coli ATCC Lysate

35 31 artifact induced during the preparation of the specimen for electron microscopy or it may be an empty area within the vesicle that has taken up phosphotungstic acid. Most of the vesicles examined consisted of granular material. In high resolution electron micrographs the internal structure of the vesicles was "beeswax" in appearance and consisted of numerous smaller sub-units equally distributed throughout the vesicle. Figure 5 is an electron micrograph of a specimen from a three week old broth culture containing bacteriophage and Escherichia coli ATCC This vesicle, unlike the vesicle in Figure 4, is not surrounded by thick "cell wall"" material. One bacteriophage is attached to the surface of the vesicle. A base plate is visible at the end of the bacteriophage tail, and a tail spike can be seen extending from the base plate to the surface of the vesicle. The tail of another bacteriophage that is apparently missing its head is also attached to the surface of the vesicle. A smaller vesicle is included within the larger one. It may be argued that the smaller vesicle is not within but on the surface of the larger vesicle. This may be discouraged by the failure to find bacteriophages attached to "inner" vesicles throughout the course of this work. It has already

36 32 ; > ^ V Fig. 5 Vesicle from Escherichia coli ATCC Lysate

37 33 been mentioned that vesicles as small as 0.1 p, have been seen. It is necessary to mention that structures smaller than 0.1 jo. have been seen attached to the tail of bacteriophage T4. These may have been analogous to the bacterial receptors attached to the tails of bacteriophage T5 (1). Magnification of the structures in Figure 5 is 220,000 times. In Figure 6 two bacteriophages and one vesicle are presented. One bacteriophage is attached to the surface of the vesicle with its tail in the contracted state. The other bacteriophage is not attached to the vesicle and is apparently entangled by the pili extending from the interior of the vesicle. A head, tail, and tail spikes are clearly visible in the unattached bacteriophage; the base plate and tail fibers are more difficult to see. The unattached bacteriophage does not represent a typical bacterial virus since its head and tail have been dislocated. Two tail fibers from the base plate of the attached bacteriophage are not in contact with the surface of the vesicle. Due to the low contrast of other micrographs it has not been possible to determine if bacteriophage tail fibers were attached to the surface of other vesicles. If bacteriophage tail fibers were not necessary as Figure 6 indicates, phages may have remained attached to vesicles by means of their tail

38 34 S ' 'V- 3 r,.: "vijsssiis^ 'i*v. A : &dss&..-;.«*3» ~*Mi iww ' X# 1 J ijl v sn* m:mr ' w HuHngBg :*>: v- *?-»'«j&jjlg OTPi 3f ^SraBL! ' V _ -' f wl.^"hkh Fig. 6 Vesicle from Escherichia coli ATCC Lysate

39 35 core or tail spikes. Although the tail spikes are below the resolution of the micrograph in Figure 6, a tail core may be seen penetrating the surface of the vesicle. The surface of the vesicle around the attached bacteriophage is altered more than the surface around the remainder of the vesicle (compare with Figure 4). The intermittent fragments of white material appearing in the surface of the vesicle might suggest the presence of cell wall material. Figure 6 is an electron micrograph of a specimen taken from a two week old broth culture of Escherichia coli ATCC and bacteriophage T4. Magnification is 220,000 times. Figure 7 is an electron micrograph of a specimen removed * from a two and one half week old broth culture of Escherichia coli ATCC containing a bacteriophage isolated from Denton, Texas sewage. Magnification is 210,000 times actual size. "Vesicles within vesicles" have been seen repeatedly in lysozyme, penicillin, and bacteriophage lysates. This electron micrograph suggests that the small spherical vesicle has been released from the larger one. The larger vesicle is surrounded by the thick "cell wall" material revealed in previous micrographs (Figures 2, 4).

40 36 tth&* -. *' "ft Fig. 7 Vesicle from Escherichia coli ATCC Lysate

41 37 It is possible that the material presented in this micrograph is analogous in origin to the vesicles "budding" from the surface of the bacterial ceil presented in Figure 2. Some of the vesicles presented in Figure 2 contain smaller structures, It is reasonable to assume that the vesicles released from the surface of a disrupted cell are capable of releasing smaller vesicles. The vesicles occurring in Figures 2 and 7 are from the same bacterial strain. It is of interest to note that only the vesicles produced in lysates of Escherichia coli ATCC retain the thick "cell wall" material. This organism was not selected for lysozyme lysis because of its high resistance to lysis by this enzyme. Escherichia coli ATCC was readily lysed by treatment with lysozyme. It is only suggested that a relationship exists between the retention of the "cell wall" and the resistance to lysozyme lysis. It is necessary to compare the thickness of the "cell wall" of Escherichia coli ATCC in Figure 1 to that of Escherichia coli ATCC in Figure 2. Figure 8 is an electron micrograph of a specimen removed from a 48-hour broth culture of Escherichia coli ATCC and bacteriophage T4. Magnification is 440,000 times. The vesicle is not typical of the vesicles usually seen in lysates of this organism since it appears to include five vesicles,

42 38 V Fig. 8 Vesicle from Escherichia coli ATCC Lysate

43 39 two of which are bacteriophage T4. Other vesicles appearing in T4-phage lysates also contained T4 phages but their occurrence was infrequent. It is suggested in the "Discussion" of this paper that spherical vesicles might be a stage in the development of bacteriophages. Since the literature has not reported the presence of bacteriophages in thin sections of vesicles, it may be argued, as in Figure 5, that the phages are on the surface of the vesicle. It is possible that the three remaining structures on the vesicle are bacteriophages "standing on their tails.,r If the phages were above the surface of the vesicle they would not be in the same plane of focus as the rest of the material in this micrograph. The initial magnification on the electron microscope was 40,000 times. If the bacteriophages were above the surface of the vesicle and out of the plane of focus, they would probably not be as distinct as the bacteriophages with tails. Figure 9 represents multiple bacteriophage attachment to the surface of a vesicle. The specimen wus removed from a three-week broth culture containing bacteriophage and Escherichia coli ATCC and is magnified 354,000 times. Multiple bacteriophage attachment was frequently observed in * bacteriophage lysates removed from broth cultures.

44 40. \. H JUf" *"Vv Fig. 9 Vesicle from Escherichia coli ATCC Lysate

45 CHAPTER BIBLIOGRAPHY 1. Weidel, W. and Kellenberger, E., "The Escherichia coli B Receptor for Phage T5," Biochiraica et Biophsica Acta, XVII (1955), 1-9. AT

46 CHAPTER IV DISCUSSION Many bacteria possess "membranous organelles" or "vesicles" in their cytoplasm; however, the origin of these vesicles remains obscure. If all membranous vesicles that have been reported in bacteria occur within intact cells and are released during physical, chemical, or mechanical disruption, they should be detected in thin sections of untreated cells. This is not the case in all bacteria. "Mitochondria-like" organelles have been observed in thin sections of intact cells of Mycobacterium avium, Mycobacterium thamnopheos, and Mycobacterium tuberculosis var. hominis (9, 12, 15). Fitz-James (4) has reported "perisporal" membranous organelles connecting the spore envelope and cytoplasmic membrane in Bacillus species. Clauert and Hopwood (5) have described extensive membranous components in the cytoplasm of Streptomyces coelicolor that are converted into "round vesicles" following destruction of the hyphae. The membranous vesicles occurring in intact cells of Micrococcus lysodeikticus have been isolated by Salton and

47 43 Chapman (14) by lysing the cells with iysozyme. Smith (16) has reported "hollow vesicles" in methionine-deficient auxotrophs of Escherichia coll; similar vesicles have been reported in thin sections of Escherichia coli stained with lanthanum (1). In thin sections of Rhodospirillum rubrum numerous vesicles (chromatophores) are visible in the cytoplasm as discrete structures (19). The cytoplasmic contents of Azotobacter agilis may be removed by mechanically shaking the cells in the presence of glass beads. Electron-microscopic examination of the disrupted cells reveals numerous spherical vesicles, some of which appear to originate from the ceil membrane (13). In cells escaping mechanical disruption, the vesicles are either not present or are undetected due to the density of the intracellular material. Mercer (11) has reported that the vesicles appearing in T2-phage lysates may contain attached T2 phages. Since T2- phage receptor sites reside in the cell wall (23), Cota-Robles (2) suggested that some of the vesicles may retain cell wall material. Another possibility exists: if,-after bacteriophage attachment, the cell wall material is destroyed, phages may remain attached to the surface of the vesicles by means

48 (tail core) other than their specific attachment organs (tail fibers). It has been shown by Kellenberger et ajl. (8) and others (24, 25) that the tail fibers of bacteriophage T4 are necessary for phage attachement to the host cell. In an electron micrograph presented in this report (Figure 6), the tail fibers of one phage are not involved with the attachment of the phage to the surface of a vesicle. If cell wall material is not present, the vesicles appearing in phage lysates may be "subprotoplasts" limited only by cell membranes. It is not suggested that subprotoplasts are viable (like true protoplasts of bacterial cells), but they may retain biochemical activities comparable to the activities in vesicles reported by other workers. Weibull et al. (22) and Weibull and Beckman (21) have shown that particles isolated from stable Proteus L cultures have a definite ultrastructure. The particles had a diameter of 0.1 to 0.4 jli and consisted of granular elements (presumably ribosomes) that were enclosed by a "peripheral unit,membrane." Biochemical studies revealed that the particles contained ribonucleic acid and some enzymes (catalase and succinic dehydrogenase); however, they contained little or no deoxyribonucleic acid and exhibited no measurable growth. 6

49 45 Weibull (20) and Storck and Wachsraan (17) have shown that "ghosts" of Bacillus meg-ateriuin contain cytochrome enzymes responsible for succinate, a-ketoglutarate, lactate, and malate oxidation. Guillaume et a1. (6) observed pleomorphic "vesicles within vesicles" m lysates of Escherichia coli following treatment of intact cells with digitonin. Biochemical activities have been linked to these vesicles. Since some vesicles in T2-phage lysates appear as incomplete spherical units, Mercer (11) has suggested that the vesicles are formed by "the rounding-up of lysed bacterial membranes." No distinction was made between bacterial cell walls and bacterial membranes. Cota Robies and Cofrman (3) have shown that the cell wall of Escherichia coli may assume a coiled configuration following lysis by T2 phage, and suggested that the vesicles were not artifacts due to lysis but were structures formed by inward loops of the cytoplasmic membrane. Two electron micrographs (Figures 1, 2) suggest that vesicles in bacteriophage T4 lysates are either formed prior to lysis or during lysis of the bacterial cell. The literature fails to report vesicles in penicillin and lysozyme lysates of Escherichia coli- The vesicles reported in this thesis are morphologically similar to

50 46 the vesicles found in T4-phage lysates; however, chemical similarity has not been determined. If the vesicles in penicillin or lysozyme lysates of Saeherichia coli are limited by cell wall material, bacteriophage titers should be reduced by their presence. Further investigation is necessary to support this concept. The only evidence which might support Wyckoff's (26) concept that spherical structures are a stage in the synthesis of bacteriophages is the vesicle presented in Figure 9. Ta* infrequent occurrence of vesicles containing bacteriophages discourages this concept. Whether the vesicle contains the two bacteriophages or whether the phages are on the surface of the vesicle cannot be determined with any accuracy by this preparation. The presence of bacteriophages in thin sections would highly suggest that phages are included within the vesicles. Wyckoff's (26) shadow-casted preparation revealed phage contact with the outer perimeter, not within or on the surface of a vesicle. Further investigation is necessary to determine the origin of the vesicles appearing in lysates of Escherichia coli. Whether the vesicles are membrane and/or ceil wall in origin cannot be determined from the information presented in this paper.

51 47 Fitz-James (4) has given the term "mesosome" to invaginated growths of the plasma membrane. Since mesosomes in Escherichia coli have been reported (7, 10, 10), the possibility 'exists that some of the vesicles presented in this work are analogous in origin to the mesosomes reported in intact cells.

52 CHAPTER BIBLIOGRAPHY 1. Caro, L. G., Van Tubergen, R. P., and Forro, F., "The Localization of Deoxyribonucleic Acid in Escherichia coli/" Journal of Biophysical and Biochemical Cytology,, IV (1958), Cota-Robles, E. K., "Electron Microscopy of Lysis from Within of Escherichia co1i by Coliphage T2," Journal of Ultrastructure Research, XI {1964), Cota-Robles, E. H. and Coffman, M. D., "Electron Microscopy of Lysis from Without of Escherichia coli B by Coliphage T2," Journal of Ultrastructure Research, X (1964), Fitz-Jaraes, P., "Participation of the Cytoplasmic Membrane in the Growth and Spore Formation of Bacilli," Journal of Biophysical and Biochemical Cytology, VIII (1960), Glauert, A. and liopv/cod, D., "The Fine Structure of Streptomyces coelicolor. I. The Cytoplasmic Membrane System," Journal of Biochemical and Biophysical Cytology, VII (1960), Guillaume, J., Francois, I)., Petitprez, A., Derieux, Jean-Claude, Palmont, J., and Nisman, 3., "Biologie Molecularire Etude au Microscope Electronique de Fractions Particulees d 'Escherich.1a coli," Compt.es Rendus de L'academle des Sciences, CCLXII (1966), Kay, J. J. and Chapman, G. B. "Cytological Aspects of Antimicrobial Antibiosis. III. Cytologically Distinguishable Stages In Antibiotic Action of Colistin Sulfate on Escherichia coli, Journal of Bacteriology, LXXXVI (1963),

53 45 8. Kellenberger, A., Bolle, 3., Epstein, N. C. P., Jerne, N. K., Reale-Scafati, A., and Sechaud, J., "Functions and Properties Related to the Tail Fibers of Bacteriophage T4," Virologyj, XXVI (.1965), Koike, M. and Takeya, K., "Fine Structure of Intracytoplasmic Organelles of Mycobacteria, 11 Journal of Biophysical and Biochemical Cytology, IX (1961), Kushvarev, V. M. and Pereverzev, N. A., "The Membranes in Escherichia coli Cells, " -Journal of Ultrastructure Research, X (1964), Mercer, E., "An Electron Microscopic Study on Thi/i Sections of Bacteria and Bacteriophage Grown on Agar Plates,"' Biochimica et Biophysica Acta, XXXIV (1959), Mudd, S., Winterscheid, C., DeLamater, 3... and Henderson, J., "Evidence Suggesting that the Granules of Mycobacteria are Mitochondria," Journal of Bacteriology, LXII (1951), Pangborn, J., Marr, A. G., and Robrish, S. A., "Location of Respiratory Enzymes in Intracytoplasmic Membranes of Azotobacter agilis," Journal of Bacteriology, LXXXIV (1962), Salton, M. R. J. and Chapman, J. A., "Isolation of the Membrane-Mesosome Structures from Micrococcus lysodeikticus," Journal of Ultrastructure Research, VI (1962), Shinohara, C., Fukushi, K., and Suzuki, J., "Mitochondrialike Structures in Ultrathin Sections of Myccbacteriurn avium," Journal of Bacteriology, LXXIV (1957), Smith, K. R. ; > "An Electron-Microscopic Study of Methionine Deficient Escherichia, colit " Journal of Ultrastructure Research, IV (1960), Storck, R. and Wachsnian, J. T., "Enzyme Localization in Bacillus megatheriura, " Journal of Bacteriology, LXXIII "(1957),

54 og 18. Vanderwinkel, S. and Murray,, R. G. E., "Organelles Intracytop1asmiqucs Bacteriens et Site d'activite Oxydo-Reductrice," Journal of Ultrastructure Research, VII (1962), Vatter, A. S. and Wolf, R. S., "The Structure of Photosynthetic Bacteria, " Journal of.bacteriology, LXXV (1958), Weibull, C., "Characterization of the Protoplasmic Constituents of Bacillus megatherium," Journal of Bacteriology, LXVI (1953), Weibull, C. and Beckman, K-, "Chemical and Metabolic properties of Various Elements Pound in Cultures of a Stable Proteus L Form, 51 Journal of General Microbiology, XXIV (1961), Weibull, C., Mohri,. T. and Afzelius, B. A., "The Morphology and Fine Structure of Small Particles in Cultures of a Proteus L Form," Journal of Ultretstructure Research, XII (1965), Weidel, W. and Primgosigh, J., "Biochemical Parallels Between Lysis by Virulent Phage and Lysis by Penicillin," Journal of General Microbio1ogy, XVIII (1958), Wildy, P. and Anderson, T. F., "Clumping of 3useeptable Bacteria by Bacteriophage Tail Fibers," Journal of General Microbiology, XXXIV (1964), Williams, R. C. and Frazer, D., "Structural and Functional Differentiation in T2 Bacteriophage," Virology, II (1956), Wyckoff, R., "Possible Imature Forms of Bacteriophage," Experentia, VIII (1950),