electron microscopy of vesicles present in bacterial …/67531/metadc163896/... ·...

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

of the Graduate S c h o o l \ Dean

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

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

i n

LIST OP ILLUSTRATIONS

Figure Page

1. Lysis of Escherichia coli ATCC 11303 24

2. Lysis of Escherichia coli ATCC 11775 . . . . . . 26

3. Lysis of Escherichia coli ATCC 11303 by Lysozyme 28

4. Vesicle from Escherichia coli ATCC 11775 Lysate 30






IV

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 appar-

ently 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

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 compo-

sition 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 suscepti-

bility to bacteriolytic agents. Although the cell walls of

Gram-positive and Gram-negative bacteria consist of essen-

tially 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).

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 lipo-

protein 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 lipo-

protein and lipopolysaccharide are responsible for bacterio-

phage attachment. The inner mucopolymer complex is responsible

for the structural rigidity of the bacterial cell; its pres-

ence 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 bac-

teriophages are well known bacteriolytic agents of biological

origin and are capable of lysing susceptible bacteria by

partial or total destruction of the mucopolymer complex.

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.

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 com-

pletely devoid of cell wall material. They may occur

spontaneously in bacterial cultures (29) or be induced in

the laboratory by treating normal cells (usually Gram-

positive 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 re-

production; 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."

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 proto-

plasts 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. Klieneberger1s

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). Bac-

terial 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 sphero-

plasts (9), are capable of reverting to normal cells.

Hofschneider (13) has applied the term spheroplast to

osmotically sensitive forms of Escherichia coli since these

spherical forms, unlike the protoplasts of Gram-positive bac-

teria, retain some of their cell wall material after treatment

with cell wall degrading agents.

Spheroplasts are capable of growth and division in hyper-

tonic media and revert to normal cells in the absence of the

inducing agent.

Lederberg and Clair (19) have suggested that penicillin-

induced 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 bac-

teriophages to spheroplasts of Escherichia coli. The

spheroplasts were induced by treating normal cells with

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 in-

adequate 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 phage-

lysed cultures of Escherichia coli.

9

Wyckoff (39) observed spherical granules in bacterio-

phage 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 intra-

cellular 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 concen-

tric rings composed of triple-layered membranes.

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 vesi-

cles 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.

CHAPTER BIBLIOGRAPHY

1. Berger, L. R. and Weiser, R. S., "The B-Glucosaminidase Activity of Egg-White Lysozyme," Biochimica et Biophysica Acta, XXVI (1957), 517-521.

2. Brenner, S. and Home, R., "Negative Staining Method for High-Resolution Electron Microscopy of Viruses," Biochimica et Biophysica Acta, XXXIV (1959), 103-110.

3. Brenner, S. and Stent, G. S., "Bacteriophage Growth in Protoplasts of Bacillus megatherium," Biochimica et Biophysica Acta, XVII (1955), 473-475.

4. Cary, W. F., Spilman, W., and Baron, L. S., "Protoplast Formation By Mass Absorption of Inactive Bacteriophage, Journal of Bacteriology, LXXIV (1957), 543-544.

5. Chatterjee, B. R. and Williams, R. P., "Cytological Changes in Aging Bacterial Cultures," Journal of Bacteriology, LXXXIV (1962), 340-344.

6. Cota-Robles, E. H., "Electron Microscopy of Lysis from Within of Escherichia coli by Coliphage T2," Journal of Ultrastructure Research, XI (1964), 112-122.

7. Cota-Robles, E. H. and Coffman, M. D., "Electron Microscopy of Lysis from Without of Escherichia coli," Journal of Ultrastructure Research, X (1964), 304-316.

8. Delbruck, M., "The Growth of Bacteriophage and Lysis of the Host," Journal of General Physiology, XXIII (1940), 643.

9. Diena, B. B., Wallace, R., and Greenburg, L., "The Production and Properties of Salmonella typhi Spheroplasts," Canadian Journal of Microbiology, X (1964), 543-549.

11

12

10. Fitz-James, P. C., "Cytological and Chemical Studies of the Growth of Protoplasts," Journal of Biophysical and Biochemical Cytology, IV (1958), 257-266.

11. 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), 319-338.

12. Hirokawa, H., "Biochemical and Cytological Observations During the Reversing Process from Spheroplast to Rod-Form Cells in Escherichia coli," Journal of Bacteriology, LXXXIV (1962), 1161-1168.

13. Hofscheider, P. EL, "Ti and Lambda Phage Adsorption on Protoplast-Like Bodies of Escherichia coli," Nature, CLXXXVI (1960), 568-569.

14. Klieneberger, E., "The Natural Occurrence of Pleuropneumonia-Like Organisms in Apparent Symbiosis with Streptobacillus moniliformis and other Bacteria," Journal of Pathogenic Bacteriology, XL (1935), 93-105.

15. Klieneberger-Nobel, E., "Filterable Forms of Bacteria," Bacteriological Reviews, XV (1951), 77-103.

16. Koch, G. and Dryer, W. J., "Characterization of an Enzyme of Phage T2 as a Lysozyme," Virology, VI (1958), 291-293.

17. Kohn, A., "Lysis of Frozen and Thawed Cells of Escherichia coli by Lysozyme, and their Conversion into Spheroplasts," Journal of Bacteriology, LXXIX (1960), 697-706.

18. Lederberg, J., "Bacterial Protoplasts Induced by Penicillin," Proceedings of the National Academy of Science, XLII (1956), 574-577.

19. Lederberg, J. and Clair, J., "Protoplasts and L-Type Growth of Escherichia coli," Journal of Bacteriology, LXXV (1958), 143-160.

20. Martin, H. H., "Bacterial Protoplasts-A Review," Journal of Theoretical Biology, V (1963), 1-34.

13

21. Mercer, E., "An Electron Microscopic Study on Thin Sections and Bacteriophage Grown on Agar Plates," Biochimica et Biophysica Acta, XXXIV (1959), 84-89.

22. Mitchell, P. D. and Moyle, J., "Autolytic Release and Osmotic Properties of Protoplasts from Staphylococcus aureus," Journal of General Microbiology, XVI (1963), 184-194.

23. Nermut, M. V. and Svoboda, A., "Reversion of Spheroplasts Produced by Lysozyme into Rods in Proteus vulgaris," Nature, CXCIII (1962), 396-397.

24. Perkins, H. R., "Chemical Structure and Biosynthesis of Bacterial Cell Walls," Bacteriological Reviews, XXVII (1963), 18-55.

25. Plapp, R. and Kandler, 0., "Zur Wirkungsweise Zellwandhemmender Antibiotica bei Gram-negative Bakteriem," Archiv fur Mikrobiologie, L (1965) 171-193.

26. Rogers, H. J. 'and Mandelstam, J., "Inhibition of Cell-Wall-Mucopeptide Formation in Escherichia coli by Benzlpenicillin and 6-[D(-)-alpha-aminophenylace-tamido] penicillanic acid (ampicillin) ,,r Biochemical Journal, LXXXIV (1962), 299-302.

27. 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), 512-523.

28. Stent, G. S., Molecular Biology of Bacterial Viruses, San Francisco, W. H. Freeman and Company, 1963.

29. Stolp, H. and Starr, M. P., "Bacteriolysis," Annual Review of Microbiology, XV (1965), 79-104.

30. 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), 3263-3268.

14

31. 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), 412-413.

32. Weibull, C., "The Isolation of Protoplasts from Bacillus megatherium by Controlled Treatment with Lysozyme," Journal of Bacteriology, LXVT (1953), 688-695.

33. 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), 158-166.

34. Welsh, M., "Lysis By Agents of Microbial Origin," Journal of General Microbiology, XVIII (1958), 491-497.

35. Williams, R. C. and Wyckoff, R., "Applications of Metallic Shadow-Casting to Microscopy,,r Journal of Applied Physics, XVII (1946), 23-33.

36. Work, E., "The Mucopeptides of Bacterial Cell Walls, A Review," Journal of General Microbiology, XXV (1961), 167-189.

37. Work, E. and Dewey, D. L., "The Distribution of Diaminopimelic Acid Among Various Microorganisms," Journal of General Microbiology, IX (1953), 394-406.

38. Wyckoff, R., "The Electron Microscopy of Developing Bacteriophage," Biochimica et Biophysica Acta, II (1948), 27-37.

39. Wyckoff, R., "Possible Immature Forms of Bacteriophage," Experentia, VIII (1950), 298-299.

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 ob-

served at a later time on the same day.

Growth media, glassware, and distilled water were auto-

claved 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 11303 and ATCC 11775. Bacterio-

phage T4 was obtained from North Texas State University and

was capable of lysing strain ATCC 11303. A bacteriophage

capable of lysing strain ATCC 11775 was isolated from Denton,

Texas, sewage.

15

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 11303 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 11303 and the

other contained ATCC 11775. The controls were prepared ac-

cording 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 con-

tained 0.1 ml of an 18 to 24 hour culture of the organism

and 0.1 ml of the specific bacteriophage dilution. Ten (ten-

fold) 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.

17

After 24 hours of incubation, the plates were removed

from the incubator and examined- Petri plates with well iso-

lated 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 dur-

ing the incubation period at room temperature.

Lysis By Lysozyme

A lysozyme-tris buffer-EDTA system was used to lyse

ATCC 11303 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

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 11303 to the spheroplast stage according to the procedure

of Lederberg (2). Complete lysis was effected by resuspending-

the 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 for-

mation, 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 electron-

microscopic examination. Controls were prepared according to

the procedure above, but did not contain penicillin.

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 2 3° 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 carbon-

covered 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

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 proce-

dure 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 with-

drawn 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.


1. Brenner, S. and Home, R. w., "Negative Staining Method for High-Resolution Electron Microscopy of Viruses," Biochirnica et Biophysica Acta, XXXIV (1959) , 103-110.

2. Lederberg, J., "Bacterial Protoplasts Induced by Penicillin," Proceedings of the National Academy of Science, XLII (1956) , 574-577 .

3. Repaske, R., "Lysis of Gram-Negative Bacteria By Lysozyme," Biochirnica et Biophysica Acta, XXII (1956) , 189-191.

21

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 dis-

tinctions 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

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 dur-

ing 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 se-

quence 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 11303

during bacteriolysis by bacteriophage T4. The specimen was

taken from a plaque area after 24 hours of growth at room tem-

perature, and is magnified 198,000 times.

24

W-

A

Fig. 1—Lysis of Escherichia coli ATCC 11303

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 ex-

tended 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 repre-

sent extensive destruction or alterations of the cell wall and

membrane.


during bacteriolysis. The specimen was removed from a plaque

area after 48 hours of growth at room temperature. The bac-

teriophage 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.

26

Fig. 2—Lysis of Escherichia coli ATCC 11775

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.


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

28

Li p.:.; .

i-fi r

Fig. 3—Lysis of Escherichia coli ATCC 11303 by Lysozyme

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 11775 and a bac-

teriophage are presented in Figure 4. This specimen was

removed from a three week old broth culture and is magni-

fied 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

30

. m. < iiS:j^'A StSSf3Bimfr.?*i M^c" •S&.&**SV>V* U£ t

I , - ii I fcfffe Wr • f S.r.£&&*' -. iH $t r>?/?tV':v-'.&£&*;.. VifeeMji

fo. it .'feV'; I £S^wMra& -mm £;.-& ••*

mmm

.

M S "

jv.ji * > , • > .

;';«,:;Kir,:;

• •

* $ &

>i'¥%V v- '- * -•' ?

'•< :y:S'.% BS3PI H • - .. -

v.:;

# A S # «

Fig. 4—Vesicle from Escherichia coli ATCC 11775 Lysate

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 11775. 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 bac-

teriophage 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

32

; > ^ V ••


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 pre-

sented. One bacteriophage is attached to the surface of the

vesicle with its tail in the contracted state. The other bac-

teriophage 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 bac-

teriophage 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 at-

tached 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

34

S ' 'V-

• 3 r

•,.: " v iJsSsiis

' i*v. A: &dSs&

•

.... .-;.«*3 »• • iww ~*Mi '

J • X#1

ijl v

•sn*

m:mr

:*>: £v- *?-»'«

'• w

H u H n g B g

£ j&jJlg •OTPi 3f ^SraBL ! ' V •_ -'

fwl. "HKH


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 bacterio-

phage 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 11303

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 11775 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).

36

ttH&* -. *' "ft


37

It is possible that the material presented in this micro-

graph 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 11775 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 11303 was readily lysed by

treatment with lysozyme. It is only suggested that a relation-

ship 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 11303

in Figure 1 to that of Escherichia coli ATCC 11775 in Figure 2.

Figure 8 is an electron micrograph of a specimen removed

from a 48-hour broth culture of Escherichia coli ATCC 11303

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,

38

V


39

two of which are bacteriophage T4. Other vesicles appearing

in T4-phage lysates also contained T4 phages but their occur-

rence 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 11775 and is magnified 354,000 times.

Multiple bacteriophage attachment was frequently observed in *

bacteriophage lysates removed from broth cultures.

.

40

• \ . H JUf" * " V v



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

AT

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 dis-

ruption, 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 mem-

brane 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

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 cyto-

plasm 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 dis-

rupted 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

(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 micro-

graph 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 ap-

pearing in phage lysates may be "subprotoplasts" limited

only by cell membranes. It is not suggested that subproto-

plasts 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 de-

oxyribonucleic acid and exhibited no measurable growth. 6

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 pleo-

morphic "vesicles within vesicles" m lysates of Escherichia

coli following treatment of intact cells with digitonin. Bio-

chemical activities have been linked to these vesicles.

Since some vesicles in T2-phage lysates appear as in-

complete 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. Cot a—Rob i e s and Cofrman (3)

have shown that the cell wall of Escherichia coli may assume

a coiled configuration following lysis by T2 phage, and sug-

gested 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

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.

47

Fitz-James (4) has given the term "me so some" to in-

vaginated 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.


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), 491-493.

2. Cota-Robles, E. K., "Electron Microscopy of Lysis from Within of Escherichia co1i by Coliphage T2," Journal of Ultrastructure Research, XI {1964), 112-122.

3. 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), 304-316.

4. Fitz-Jaraes, P., "Participation of the Cytoplasmic Membrane in the Growth and Spore Formation of Bacilli," Journal of Biophysical and Biochemical Cytology, VIII (1960) , 507-528.

5. 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), 479-438.

6. 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.1 a coli," Compt.es Rendus de L'academle des Sciences, CCLXII (1966), 696—698 -

7. 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), 536-543.

48

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) , 419-440.

9. Koike, M. and Takeya, K., "Fine Structure of Intracytoplasmic Organelles of Mycobacteria, 11 Journal of Biophysical and Biochemical Cytology, IX (1961) , 597-607.

10. Kushvarev, V. M. and Pereverzev, N. A., "The Membranes in Escherichia coli Cells, " -Journal of Ultra structure Research, X (1964) , 610-614..

11. Mercer, E., "An Electron Microscopic Study on Thi/i Sections of Bacteria and Bacteriophage Grown on Agar Plates,"' Biochimica et Biophysica Acta, XXXIV (1959), 84-89.

12. Mudd, S., Winterscheid, C., DeLamater, 3... and Henderson, J., "Evidence Suggesting that the Granules of Mycobacteria are Mitochondria," Journal of Bacteriology, LXII (1951), 459-4-75.

13. Pangborn, J., Marr, A. G., and Robrish, S. A., "Location of Respiratory Enzymes in Intracytopla smic Membranes of Azotobacter agilis," Journal of Bacteriology, LXXXIV (1962), 669-678.

14. Salton, M. R. J. and Chapman, J. A., "Isolation of the Membrane-Me so some Structures from Micrococcus lysodeikticus," Journal of Ultrastructure Research, VI (1962), 489-498.

15. Shinohara, C., Fukushi, K., and Suzuki, J., "Mitochondria-like Structures in Ultrathin Sections of Myccbacteriurn avium," Journal of Bacteriology, LXXIV (1957), 413-415.

16. Smith, K. R.;> "An Electron-Microscopic Study of Methionine Deficient Escherichia, coli t " Journal of Ultra structure Research, IV (1960), 213-221.

17. Storck, R. and Wachsnian, J. T. , "Enzyme Localization in Bacillus megatheriura, " Journal of Bacteriology, LXXIII "(1957), 784-790.

oG

18. Vanderwinkel, S. and Murray,, R. G. E., "Organelles Intracytop1asmiqu c s Bacteriens et Site d'Activite Oxydo-Reductrice," Journal of Ultrastructure Research, VII (1962), 185-199.

19. Vatter, A. S. and Wolf, R. S., "The Structure of Photo synthetic Bacteria, " Journal of .Bacteriology, LXXV (1958), 480-438.

20- Weibull, C., "Characterization of the Protoplasmic Constituents of Bacillus meg a th er i um," Journal of Bacteriology, LXVI (1953), 696-702.

21. 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), 379-391.

22. 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 Ultret structure Research, XII (1965), 81-91.

23. Weidel, W. and Primgosigh, J., "Biochemical Parallels Between Lysis by Virulent Phage and Lysis by Penicillin," Journal of General Microbio1ogy, XVIII (1958), 513-517.

24. Wildy, P. and Anderson, T. F., "Clumping of 3useeptable Bacteria by Bacteriophage Tail Fibers," Journal of General Microbiology, XXXIV (1964), 273-283.

25. Williams, R. C. and Frazer, D., "Structural and Functional Differentiation in T2 Bacteriophage," Virology, II (1956), 289.

26. Wyckoff, R., "Possible Imature Forms of Bacteriophage," Experentia, VIII (1950), 293-299.

CHAPTER V

SUMMARY

Penicillin, lysozyme, bacteriophages, and endogenous

autolytic enzymes destroy susceptible cells of Bsch.er.lchia

coli by bacteriolysis. Electron-microscopic examination of

these lysates reveals spherical structures of approximately 0.2

to 0.6 micron in diameter. It is suggested that the structures

may be formed within the cell prior to lysis or are formed dur-

ing dissolution of the bacterial ceil. The structures, once

liberated from the ceil, may release smaller units. Since

bacteriophages may be attached to some of these structures in

phage-lysed cultures, it is suggested that the structures are

bound, at least in part, by cell wall material.

51

BIBLIOGRAPHY

Books

Stent, G. S., Molecular Biology o£ Bacterial Viruses, San Francisco, W. H. Freeman and Company, 1963.

Articles

Berger, L. R. and Weisor, R. S. , ' Activity of Egg-White Lysosyme," Biochimica et Biophysica Acta, XXVI (1957), 517-521.

Brenner, S. and Home, R. - "Negative Staining Method for nigh-Resolution Electron Microscopy of Viruses," Biochimica et Biophysics Acta, XXXIV (1359), 103-110.

Brenner, S. and Stent, G. S., "Bacteriophage Growth in Protoplasts of Bacillus megatherium," Biochimica et Biophysica Acta, XVII (1955), 473-475.

Caro, L. G., Van Tubergen, R. P., and Forro, F., "The Localization of Deoxyribonucleic Acid in Escherichia co 1.1, " Journal of Biophysical and Biochemical Cytclogy, IV (1958), 491-493./

Gary, W. F., Spilman, W., and Baron, L. 3., "Protoplast Formation By Mass Absorption of Inactive Bacteriophage," Journal of Bacteriology, LXXIV (1957), 543-544.

Chatterjee, B. R. and Williams, R. P., "Cytological Changes in Aging Bacterial Cultures," Jour nail of Bacteriology, LXXXIV (1962), 340-344.

Cota-Robles, E. H., "Electron Microscopy of Lysis from Within of Escherichia coli by Coliphage T2, " Journal of Ultrastructure Research, XI (1964), 112-122.

52

Cota-Robles, E. H. and Coffiaan, M. D., "Electron Microscopy of Lysis from Without of Escherichia coli B by Coiiphage T2, " Journal of Ultrastruct~ure Research, X (1964} , 304-316.

Delbruck, M., "The Growth, of Bacteriophage and Lysis of the Host," Journal of General Physiology,, XXIII (1940) , 643.

Diena, B. B., Wallace, R., and Greenburg, L., "The Production and Properties of salmonella typhi Spheroplasts," Canadian Journal of Microbio1cgy„ X (1964), 543-549.

Fitz-James, P. C., "Cytological and Cheraical Studies of the Growth of Protoplasts/1' Journal of Biophysical and Biochemical Cytology, IV (1958), 257-266*

Fitz-James, P., "Participation of the Cytoplasmic Membrane in the Growth and Spore Formation of Bacilli," Journal of Biophysical and Biochemical Cytology, VIII (1S60), 507-528.

Glauert, A. and Hopwood, D . " T h e Fine S •cruccure or. Streptorgyces coellcclor - 1. The Cytoplasmic Membrane System,." Journal of Biochemical and Biophysical Cytology, VII (I960), 479-488.

Guillaurne, J., Francois, D., Petitproz, A., Derieux, Jean-Claude, Pelmont, J., and Nisman, B., "Biologie Molecularire-—Etude au Microscope Slectronique de Fractions Particulees d1 Escherichia coli," Comptes Rendus de L'acaderaie des Sciences, CCLXII (1966), 696-698.

Hillier, J., Mudd, S., and Smith,. A„, "Internal Structure and Nuclei in Cells of Eseherichia coli as Shown by Improved Electron Microscopic Techniques," Journal of Bacteriology, L V U (1949), 319-338.

Hirokawa, H., "Biochemical and Cyto logical Observations During the Reversing Process from Spheroplast to Rod-Form Cells in Escherichia coli," Journal of Bacteriology, LXXXIV (1962), 1161-1158.

Hofscheider, P. E., "Ti and Lambda Phage Adsorption on Protopla. .t-Like Bodies of Escherichia coll.," Nature, CLXXXVI (1960), 568-569.

54

Kay, J. J. and Chapman, G. 33., "Cytological Aspects of Antimicrobial Antibiosis. Ill. Cytologically Distinguishable Stages in Antibiotic Action of Colistin Sulfate on Escherichia coliJournal of Bacteriology, LXXXVI (1963), 5 36-54 3.

Kellenberger, A., Bolle, E., Epstein, N. C. F., Jerne, N. K., Reale-Scafati, A., and Sechaud, J., "Functions and Properties Related to the Tail Fibers of Bacteriophage T4-," Virology, XXVI (1965), 419-440.

Klieneberger, E., "The Natural Occurrence of Pleur©pneumonia-Like Organisms in Apparent Symbiosis with Streptobaci1lus moniliformis and other Bacteria," Journal of Pathogenic Bacteriology, XL (1935), 93-105.

Klieneberger-Nobel, E., "Filterable Forms of Bacteria," Bacteriological Reviews, XV (1951), 77-103.

Koch, G. And Dryer, W. J., "Characterization of an Enzyme of Phage T2 as a Lysozyme," Virology, VI (1958), 291-293.

Kohn, A., "Lysis of Frozen and Thawed Cells of Escherichia coli by Lysozyme, and their Conversion into Spheroplasts," Journal of Bacteriology, I,XXIX (1960) , 697-706.

Koike, M. and Takeya, K., "Fine Structure of Intracytoplasmic Organelles of Mycobacteria," Journal of Biophysical and Biochemical Cytology, IX (1961), 597-607.

Kushvarev, V. M. and Pareverzev, N. A., "The Membranes in Escherichia coli Cells," Journal of Ultrastructure Research, X (1964), 610-614.

Lederberg, J., 'Bacterial Protoplasts Induced by Penicillin," Proceedings of the National Academy of Science, XLII (1956), 574-577.

Lederberg, J. and Clair, J., "Protoplasts and L-Type Growth of Escherichia coli," Journal of Bacteriology, LXXV (1958), 143-160.

Martin, H. H. , "Bacterial Protoplasts—A Review, " Journal of Theoretical Biology, V (1963), 1-34.

55

Mercer, E., "An Electron Microscopic Study on Thin Sections and Bacteriophage Grown on Agar Plates," Biochimica et Biophysica Acta, XXXIV (1959), 34-89.

Mitchell, P. D. and Moyle, J., "Autolytic Release and Osmotic Properties of Protoplasts from Staphylococcus aureus," Journal of General Microbiology, XVI (1963), 184-194.

Mudd, S., Winter scheid f C. , DeLaraater, E., and Kendersen, J., "Evidence Suggesting That the Granules of Mycobacteria are Mitochondria," Journal of Bacteriology, LXII (1951), 459-475.

Nermut, M. V. and Svoboda, A., "Reversion of Spheroplasts Produced by Lysozyme into Rods in Proteus vulgaris," Nature, CXCIII (1962), 396-397.

Pangborn, J., Marr, A. G. , and Robrish, S. A., ""Location of Respiratory Enzymes in Intracytoplasmic Membranes of Azotobacter agilis," Journal of Bacteriology, LXXXIV (1962), 669-678.

Perkins, Ii. R., "Chemical Structure and Biosynthesis of Bacterial Cell Wails," Bacteriological Reviews, XXVII (1963), 18-55.

Plapp, R. and Kandler, 0., "Zur Wirkungsweise Zellwandhemmender Antibiotica bei Gram-negative Bakterien," Arch.lv fur Mikrobiologie, L (1965) 171-193.

Repaske, R. , "Lysis of Gram-Negative Bacteria 3y Lysozyme," Biochimica et Biophysica Acta, XXII (1956), 189-191.

Rogers, II. J. and Mandelstam, J., "Inhibition of Cell-Wall-Mucopeptide Formation in Escherichia coli by Benzl-penicillin and 6-[D(-)-alpha-aminophenylacetamido] penicillanic acid (ampicillin) , " Biochemical Journal, LXXXIV (1962), 299-302.

Salton, M. R. J., "Bacterial Cell Wall. IV. Composition of the Cell Wails of some Gram-Positive and Gram-Negative Bacteria," Biochimica et Biophysica Acta, X (1953), 512-523.

56

Salton, M. R. J. and Chapman, J. A., "Isolation of the Membrane-Mesosome Structures from Micrococcus lysodeikticus," Journal of Ultrastructure Research, VI (1962), 489-498.

Shinohara, C., Fukushi, K., and Suzuki, J., "Mitochondria-like Structures in UTtrathin Sections of Mycobacberium avium," Journal of Bacteriology, LXXIV (1957), 413-415.

'Smith, K. R., "An Electron-Microscopic Study of Methionine Deficient Escherichia coli," Journal of Ultrastructure Research, IV (1960), 213-221.

Stolp, H. and Starr, M. P., "Bacteriolysis," Annual Review of Microbiology, XV (1965), 79-104.

Storck, R. and Wachsrnan, J. T., "Enzyme Localization in Bacillus megatherium," Journal of Bacteriology, LXXIII (1957), 784-790.

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), 3263-3268.

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), 4-12-413.

Vanderwinkel, E. and Murray, R. G. E„, "Organelles Intracyto-plasmiques Bac e'er i ens et Site d ' Activite Oxydo-Reductrice, " Journal of Ultrastructure Research, VII (1962), 185-199.

Vatter, A. E. and Wolf, R. S., "The Structure of Photosynthetic Bacteria," Journal of Bacteriology, LXXV (1958), 480-488.

Weibull, C., "Characterisation of the Protoplasmic Constituents of Bacillus megatherium," Journal of Bacteriology, LXVI (1953), 696-702.

Weibull, C., "The Isolation of Protoplasts from Bacillus megatherium by Controlled Treatment with Lysozyme," Journal of Bacteriology, LXVI (1953), 688-695.

57

Weibull, C. and Beckman, H., "Chemical and Metabolic Properties of Various Elements Found in Cultures of a Stable Proteus L Form," Journal of General Microbiology, XXIV (1961), 379-391.

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 Ultrastructure Research, XII (1965), 81-91.

Weidel, W. , Frank, H., and Martin, H. H., "The Rigid Layer of the Cell Wail of Escherichia coli Strain B," Journal of General Microbiology, XXII (1960), 158-166.

Weidel, W. and Kellenberger, E., "The Escherichia coli B Receptor for Phage ?5," Biochirnica et Biophysica Acta, XVII (1955), 1-9.

Weidel, W. and Primgosigh, J., "Biochemical Parallels Between Lysis by Virulent Phage and Lysis by Penicillin,ir

Journal of General Microbiology, XVIII (1958), 513-517.

Welsh, M., "Lysis By Agents of Microbial Origin," Journal of General Microbiology, XVIII (1958), 491-497.

Wildy, P. and Anderson, T. F., "Clumping of Susceptible Bacteria by Bacteriophage Tail Fibers," Journal of General Microbiology, XXXIV (1964) , 273-283.

Williams, R. C. and Frazer, D., "Structural and Functional Differentiation in T2 Bacteriophage," Virology, II (1956), 289.

Williams, R. C. and Wyckoff, R., "Applications of Metallic Shadow-Casting to Microscopy," Journal of Applied Physics# XVII (1946), 23-33.

Work, E., "The Mucopeptioes of Bacterial Cell Walls, A Review," Journal of General Microbiology, XXV (1961), 167-189.

Work, E. and Dewey, D. L., "The Distribution of Diaminopimelic Acid Among Various Microorganisms," Journal of General Microbiology, IX (1953) 394-406.

58

Wyckoff, R., "Possible Immature Forms of Bacteriophage," Experentia, VIII (1950), 293-299.

Wyckoff, R., "The Electron Microscopy of Developing Bacteriophage," Biochimica et Biophysica Acta, II (1948), 27-37.

electron microscopy of vesicles present in bacterial …/67531/metadc163896/... ·...

Documents