Group Title: anatomy of the cell envelope of a marine vibrio examined by freeze-etching /
Title: The Anatomy of the cell envelope of a marine vibrio examined by freeze-etching /
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Title: The Anatomy of the cell envelope of a marine vibrio examined by freeze-etching /
Physical Description: 105 leaves : ill. ; 28 cm.
Language: English
Creator: Crawford, Jack Thornton, 1945-
Publication Date: 1973
Copyright Date: 1973
 Subjects
Subject: Cytology -- Research   ( lcsh )
Bacterial cell walls   ( lcsh )
Microbiology thesis Ph. D
Dissertations, Academic -- Microbiology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1973.
Bibliography: Includes bibliographical references (leaves 99-104).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Jack Thornton Crawford.
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Bibliographic ID: UF00097573
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000566393
oclc - 37869450
notis - ACZ2825

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THE ANATOMY OF THE CELL ENVELOPE OF A MARINE VIBRIO
EXAMINED BY FREEZE-ETCHING







By




JACK T. CRAWFORD


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY






UNIVERSITY OF FLORIDA
1973














ACKNOWLEDGMENTS


I wish to express my appreciation to the following people whose

efforts made this study possible.

I wish to thank Dr. Max E. Tyler for his guidance, encouragement,

and patience during the course of this study.

I wish to thank Dr. Henry C. Aldrich for his considerable efforts

in my training in all aspects of electron microscopy, for the use of

the facilities of the Biological Ultrastructure Laboratory, and for his

encouragement and friendship.

I wish to thank Dr. Arnold S. Bleiweis for serving on my advisory

committee and for the use of his facilities in preparing the amino acid

analyses.

I wish to thank Dr. Daniel Billen for serving on my advisory

committee.

I wish to especially thank my wife, Kay, for her unfailing under-

standing and patience.















TABLE OF CONTENTS



Page
ACKNOWLEDGMENTS . . . . . . . . . . . ii

LIST OF TABLES . . . . . . . . . . iv

LIST OF FIGURES . . ,. . . . . . . . v

KEY TO SYMBOLS . . . . . . . . . . .ix

ABSTRACT . . . . . . . . . . . . x

INTRODUCTION . . . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . .. .. . 13

RESULTS . . . . . . . . ... . . . . 25

DISCUSSION . . . . . . . . . ...... 89

LITERATURE CITED . . . . . . . . ... . 93

BIOGRAPHICAL SKETCH . . . . . . . . . . 105














LIST OF TABLES


Table Page

1. Amino acid analysis of purified peptidoglycan . 29

2. Determination of the specificity of antibody
labelling . . . . . . . . . 78
















LIST OF FIGURES


1. Longitudinal and cross sections of cells
fixed in glutaraldehyde and Os04 . . . .

2. Cross section of a cell showing the double-
track appearance of the membranes . . .

3. Cross section of a cell showing an extra layer
inside the cytoplasmic membrane . . . .

4. Purified trypsin-treated peptidoglycan negatively
stained with potassium phosphotungstate . .

5. Purified LPS positively stained with uranyl
acetate . . . . . . . . . .

6. Purified LPS positively stained with uranyl
acetate . . . . . . . . .

7. Freeze-etched cells suspended in complete
salts without glycerol . . . . . .

8. Freeze-etched cell suspended in complete
salts, showing the fracture face of
the cytoplasmic membrane . . . . .


27


27


S 27


S 31


34


34


37


9. Unetched convex fracture of cells without
glycerol showing the smooth fracture
face of the outer membrane . . . .

10. Deep etched cell in complete salts showing
the outer surface of the cell revealed
by etching . . . . . . . .

II. Freeze-etched cell showing the smooth
convex fracture face of the outer
membrane and the cell surface . . .

12. Freeze-etched cell showing the concave
fracture face of the cytoplasmic
membrane . . . . . . . .


. . 39



. 41


. . 43


Figure


Page







Figure


Freeze-etched cell showing the smooth
concave fracture face of the outer
membrane . . . . . . . . .

Concave fracture of a cell showing a
surface with distinct subunit structure

Freeze-etched cell showing a paracrystalline
array inside the cytoplasmic membrane .

Freeze-etched glycerol-treated cell showing
the concave fracture face of the
cytoplasmic membrane . . . . . .

Freeze-etched glycerol-treated cell showing
the convex fracture face of the
cytoplasmic membrane . . . . . .


. . 43


. . 43


. . 46



. . 48


18. Freeze-etched glycerol-treated cell showing
the smooth concave fracture face of
the outer membrane . . . . . . . 50

19. Convex fracture of a cell showing the smooth
fracture face of the outer rerrbrane . . .. 50

20. Freeze-etched glycerol-treated cell showing
the rough convex fracture face of the
rigid layer . . . . . . . . 52

21. Freeze-etched glycerol-treated cell showing
the rough convex fracture face of the
rigid layer . . . . . . . ... .52

22. Freeze-etched glycerol-treated cell showing
the concave face of the globular layer . . 54

23. Freeze-etched glycerol-treated cell showing
the concave face of the globular layer ..... 54


Concave fracture of a glycerol-treated cell
in which the globular layer is
incomplete . . . . . . .

Cross fractured cell envelopes of three
cells showing the ej es of the
cytoplasmic membrane, rigid layer, and
outer membra e . . . . . . .

Freeze-etched cell showing the cross
fractured cell wall . . . . .


. . 57





. . 57


. . 57


Page









Figure


Complementary surfaces of a glycerol-
treated cell observed by double-replica
technique, showing the fracture faces
of the cytoplasmic membrane . . . ... .59

Complementary fracture faces of a glycerol-
treated ce.ll showing the concave globular
layer and the convex rough layer . . . . 61

Complementary fracture faces of a glycerol-
treated cell showing the smooth fracture
faces of the outer membrane . . . ... .63

Thin section of isolated cell envelopes prepared
by lysing bacteria in a French pressure cell 66

Thin section of isolated cell envelopes prepared
by lysing bacteria in a French pressure cell 66

Isolated cell envelopes positively stained with
uranyl acetate . . . . . . . ... .66

Freeze-etched isolated cell envelopes showing
the convex fracture face of the cytoplasmic
membrane . . . . . . . .... . .68

Freeze-etched isolated cell envelopes showing
the surface exposed by etching . . .... . 68

Freeze-etched isolated cell envelopes showing
the concave globular layer . . . . .68

Freeze-etched isolated cell envelopes showing
the globular layer exposed by etching
alone . . . . . . . ... .68


Freeze-etched cell walls. The walls were
prepared by lysing cells with Triton
X-1 0 . . . . . . . . .

Freeze-etched cell walls . . . . .

Freeze-etched cell walls . . . . .

Freeze-etched cell walls . . . . . .

Freeze-etched cell walls digested with
lysozyme, showing the fibrous concave
etch surface . . . . . . . .


S 71


S. 74


Page







Figure


Freeze-etched cell walls digested with
lysozyme, showing the fibrous concave
surface and the globular layer . . . . .


43.




44.



45.


46.



47.




48.


49.


50.




51.




52.




53.


Freeze-etched cell walls digested with
lysozyme, showing the fibrous concave
surface emerging from the ice, indicating
that it is an etch surface . . . . .

Freeze-etched isolated outer membrane material
showing the concave and convex smooth fracture
faces of the vesicles . . . . . .

Fractured outer membrane material showing the
fracture face and the unfractured surface

Thin sectioned cell labelled with ferritin-
conjugated antiserum. This cell was fixed
following the antibody treatment . . . .

Thin sectioned cell labelled with ferritin-
conjugated antiserun. This cell was
fixed in glutaraldehyde prior to anti-
body treatment . . . . . . . .

Freeze-etched unfixed cell labelled with
ferritin-conjugated antiserum . . . .

Freeze-etched unfixed cell labelled with
ferritin-conjugated antiserum . . . .


Freeze-etched unfixed cell labelled with
ferritin-conjugated antiserum showing
the fracture face of the outer
membrane . . . . . . . . . .

Freeze-etched unfixed cell labelled with
ferritin-conjugated antiserum showing
the fracture face of the outer
membrane . . . . . . . . . .

Freeze-etched glutaraldehyde fixed
ferritin labelled cell showing the
relationship of the smooth fracture face
and the ferritin labelled surface . . . .

Diagramatic representation of the cell
envelope of the marine vibrio as
determined by freeze-etching. The
location of the fracture planes are
illustrated . . . . . . . . .


viii


S 74



76


76



S 80




80


83


83


Page















KEY TO SYMBOLS


CM cytoplasmic membrane

0 outer membrane

CM cytoplasmic membrane fracture face, convex

CM cytoplasmic membrane fracture face, concave

R rigid layer fracture face, convex

G globular layer fracture face, concave

outer membrane fracture face, convex
0 outer membrane fracture face, concve
O outer membrane fracture face, concave

0 sur actual outer surface of the outer membrane

0 sur actual inner surface of the outer membrane

cyto cytoplasm

S direction of shadow







Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



THE ANATOMY OF THE CELL ENVELOPE OF A MARINE VIBRIO
EXAMINED BY FREEZE-ETCHING

By

Jack T. Crawford

December, 1973


Chairman: Max E. Tyler
Major Department: Microbiology

The structure of the cell envelope of the marine vibrio MW40 was

examined by freeze-etching and other techniques. In thin section the

organism was similar to other gram-negative bacteria. The cell enve-

lope appeared as two double-track layers. No intermediate dense layer

was observed.

Purified peptidoglycan was prepared from cells during the exponen-

tial phase of growth by treating cells with hot sodium dodecyl sulfate.

The peptidoglycan had a typical amino acid and amino sugar composition

and did not have a covalently linked lipoprotein. The material appeared

fibrous when observed in the electron microscope.

Purified lipopolysaccharide (LPS) was extracted by the hot phenol-

water procedure. Its composition and appearance was typical. The LPS

was used to determine the specificity of anti-LPS antiserum.

For freeze-etching,cells were suspended in a salt solution con-

taining 0.22M NaCI, 0.026M MgCl2, and 0.01M KC1. In some cases 20%

glycerol was added as a cryoprotective agent. The cell envelope of

this organism freeze-fractured in three planes. One fracture split the








cytoplasmic membrane along its hydrophobic center revealing particle-

studded fracture faces similar to those seen in other bacteria. The

convex face was more densely covered with particles than the concave

face. Large particle-free areas were observed on both faces. The

appearance of the faces was the same with and without glycerol.

A second fracture revealed smooth particle-free concave and convex

faces. This fracture occurred primarily without glycerol, but was

occasionally seen in glycerol-treated cells.

The third fracture produced a rough convex face and a concave face

composed of subunits approximately 10 nm in diameter. In areas where

the subunit layer was incomplete it was observed that the subunits

were globular and were backed by a smooth surface. This fracture

occurred in cells freeze-etched with glycerol, and occasionally the

globular surface was seen without glycerol.

The outer surface of the cell was exposed by etching in prepara-

tions without glycerol. The surface appeared smooth or finely granular,

but may have been obscured by a thin eutectic layer.

Complementary replicas were prepared and it was demonstrated that

the three pairs of fracture faces were in fact apposed and were produced

by three fractures.

Isolated cell envelopes were prepared by lysing cells in a French

pressure cell. The appearance of these envelopes when freeze-etched

was similar to whole cells. It was observed, however, that the globular

surface could be exposed by etching alone indicating that it had sepa-

rated from the rough surface before freezing.

Crude outer membrane material was prepared by washing the cells








with NaCl and sucrose solutions. This material freeze-fractured produc-

ing smooth concave and convex faces.

Specific rabbit anti-LPS antiserum was prepared and used to label

the LPS on the cell surface. This antibody was then labelled with

ferritin-conjugated anti-rabbit immunoglobulin antiserum. In thin

sections these cells appeared coated with a band of ferritin along

the outer double-track, but separated from it by an electron transparent

space. For freeze-etching the cells were suspended in O.05M MgCl2.

The preparations were deep etched and the presence of the ferritin on

the etch face proved that the outer surface of the cell was revealed.

The smooth convex fracture face was immediately adjacent to this ferritin

coated surface.

It was concluded that the envelope fractured at three levels. One

fracture split the cytoplasmic membrane. A second split the outer

membrane along its hydrophobic center revealing smooth fracture faces.

The third fracture exposed the rough convex surface of the rigid layer

and a globular layer which separated the rigid layer from the outer

membrane.















INTRODUCTION


The cell envelopes of gram-negative bacteria are multilayered

structures of complex composition. Numerous studies have been reported

which dealt with the chemical composition, structure, and biosynthesis

of the various components of the envelope. The organization of these

components into the functional cell envelope has also been extensively

studied. Related work has focused on the antigenic specificity of the

envelope and its role in pathogenicity. The result has been the

formulation of a generally accepted, though not particularly detailed,

model of the gram-negative cell envelope (16).

In the study reported here the structure of the cell envelope of a

marine vibrio was examined, primarily by freeze-etching. The purpose

of this investigation was first to compare the structure of this organism

with that of more commonly studied gram-negative bacteria such as

Escherichia coli. The second and main objective was to determine if

the technique of freeze-etching could be used to obtain additional in-

formation on the structure of the cell envelope of gram-negative

bacteria in general.

The cell envelope of gram-negative bacteria consists of at least

three layers, the cytoplasmic membrane, the rigid layer, and the outer

layer or membrane. The latter two layers comprise the cell wall,

although this term is often used to designate the rigid layer alone.

The cytoplasmic membrane appears to be a typical membrane composed of








phospholipid and protein, and is comparable in structure and function

to the more easily studied cytoplasmic membrane of gram-positive

bacteria (54).

The rigid layer is composed of peptidoglycan and associated pro-

teins. It is in the form of a "bagshaped macromolecule" which gives

the cell shape and strength (74). The outer membrane contains phos-

pholipid, lipopolysaccharide, and proteins or lipoproteins, and is

unique to gram-negative bacteria. The term "membrane" is used to denote

its appearance when thin-sectioned and viewed in the electron microscope,

and does not imply that it has the functions of other membranes.

In addition to this basic structure, certain bacteria have layers

external to the outer membrane. These extra layers are usually found

in halophilic, photosynthetic, and other more unusual bacteria. There

are a number of excellent reviews on the structure and composition of

the gram-negative cell envelope (14, 25, 27, 30, 34, 35, 47, 53).

Chemical composition and structure of envelope components.--Pepti-

doglycan has been isolated from a variety of gram-negative bacteria

by various methods, usually involving phenol, hot detergents, or

other hzrsh treatments, combined with enzymatic digestions. Pepti-

doglycan consists of a glycan backbone of alternating N-acetyl

glucosamine and N-acetyl muramic acid residues with a peptide moiety

linked to the carboxyl group of muramic acid. In E. coli the peptide

is composed of L-alanine, D-glutamic acid, meso-diaminopimelic acid,

and D-alanine. Some of the peptide chains are cross-linked from the

amino group of diaminopimelic acid to the carboxyl group of D-alanine.









This basic structure appears to be universal for gram-negative

bacteria, although in many cases all that is known about the pepti-

doglycan is the amino acid content (56). The amount of peptidoglycan

varies from about 10% of the cell wall to none in the case of certain

halophiles.

Braun and coworkers (10, 12, 13) have purified a peptidoglycan-

lipoprotein complex from E. coli and shown that the lipoprotein is

covalently linked to the carboxyl group of diaminopimelic acid. This

linkage is specifically split by trypsin allowing the lipoprotein to

be solubilized with hot sodium dodecyl sulfate (SDS). The amino acid

sequence of the protein has been determined, and it was found that the

lipid is covalently bound (8). The molecular weight of the lipoprotein

is about 10,000.

These workers have also reported that a similar lipoprotein is

attached to the peptidoglycan of several strains of Salmonella and

Serratia marcescens, but is not found in Pseudomonas fluorescens or

Proteus mirabilis (11). They later found that if the peptidoglycan of

P. mirabilis is isolated from stationary rather than exponential phase

cultures, a lipoprotein is attached (see ref. 36). No lipoprotein was

found in the marine pseudomonad studied by MacLeod's group (24).

Weidel et al. (73) studied metal-shadowed preparations of isolated

rigid layers of E. coli and found cell-shaped granular structures with

globular units on their surface. Digestion with proteolytic enzymes

removed these globules. Recently, Martin et al. (36) examined negatively

stained preparations of purified peptidoglycan. All of the preparations

appeared as granular cell-shaped structures. Layers from E. coli and

P. mirabilis were covered with globular particles which were about 9 to







10 nm in diameter and about 20 nm apart. Preparations from Pseudomonas

aeruginosa showed considerably fewer particles, and layers from

Spirillum serpens were free of particles. The particles on the

Pseudomonas peptidoglycan were readily removed by proteolytic enzymes.

Although it would be convenient to associate these particles with

the lipoprotein studied by Braun, they are simply too large for a

molecule with a molecular weight of 10,000. It is possible, however,

that the lipoprotein molecules occur in groups and aggregate to form

larger units.

The composition and structure of the lipopolysaccharides (LPS)

of the outer membrane have also been extensively studied (34). They

are readily isolated by the hot phenol-water extraction procedure (76)

and have been characterized in a wide variety of bacteria. The lipid

portion, lipid A, is covalently linked to a carbohydrate core contain-

ing the unusual sugars keto-deoxyoctanoic acid and glycero-D-mannoheptose.

Attached to the core are the 0-antigenic side chains, the: composition

of which varies according to the strain of bacteria. In rough strains

the side chains are short or absent. When purified LPS is observed in

the electron microscope it appears to have the structure of membranes.

Shands et al. (62) observed LPS positively stained with uranyl acetate

and found various forms, all of which had areas which appeared membrane-

like, that is as a trilaminar or double-track appearance. They also

found that the dimensions of the double-track were the same in LPS

from smooth and rough strains, indicating that the polysaccharide side

chains are not stained and are not seen in thin sections. dePetris

(16) observed that thin sectioned LPS also has a double-track appearance.

The cell envelope also contains a considerable amount of lipid,





5


with the major portion being phospholipid. The predominant fatty acids

are C16 and Cl8 straight chain acids, and -hydroxymyristate is found

in the LPS. Sterols are absent.

In most descriptions of the outer membrane lipoproteins are listed

as a major component. .The work on these molecules has been very vague

and no one has clearly shown that there are any true lipoproteins, that

is covalently linked lipid and protein, in the outer membrane.

Recently, the proteins of the cell envelope of E. coli have been

studied using SDS-polyacrylamide gel electrophoresis (57, 58).

Schnaitman found from 20 to 30 bands of protein in cell envelope

extracts, and one major protein possessing an apparent molecular weight

of 44,000. By using sucrose gradient centrifugation he was able to

partially separate the cell wall and cytoplasmic membrane. The major

protein band was found to be localized in the cell wall and accounted

for 70% of the wall protein. Although further work as indicated that

the initial results may be oversimplified, the basic conclusion, that

there are major structural proteins, is still valid (Schnaitman,

personal communication). Comparable results have been obtained in

E. coli (31) and Salmonella typhimurium (48).

Studies of proteins released from P. aeruginosa by ethylenediamine-

tetraacetic acid (EDTA) treatment indicate that structural proteins

exist in the cell wall (68). The location of these proteins will be

discussed later.

Fine structure of the cell envelope.--When thin sections of gram-

negative bacteria are examined in the electron microscope, the cell

envelope appears to consist of a smooth inner membrane and a wavy outer

membrane. Both of these membranes have a double-track appearance and







measure about 7.5 nm in width. In early work these were the only

structures seen, but improved techniques have allowed the visualiza-

tion of the intermediate rigid layer (43). This general anatomy has

been observed in many gram-negative bacteria and no attempt will be

made to review this literature (27).

Aside from its double-track appearance, the cytoplasmic membrane

does not have any fine structure detectable in thin sections. Because

of the difficulty in separating it from the cell wall, the membrane has

not been thoroughly studied by negative staining, but available results

indicate that it does not have a subunit structure (55).

The appearance of the intermediate dense layer, or rigid layer,

varies according to the organism studied, the method of fixation, and

the method of staining (27). In some bacteria this layer is seldom or

never seen. This may be due to a thinner layer of peptidoglycan (24)

or possibly to the lack of a covalently bound lipoprotein. The rigid

layer generally follows the contours of the cytoplasmic membrane. It

has a thickness of 3 to 8 nm.

Evidence that the peptidoglycan is associated with the dense layer

seen in thin sections is provided by lysozyme digestion. Cells which

have been treated with lysozyme and EDTA lack the intermediate layer,

and this treatment allows the outer layer to separate from the cyto-

plasmic membrane (16, 43). It is not known whether the metal stain

is localized in the peptidoglycan alone, or the dense layer represents

another layer which is solubilized when the peptidoglycan is digested.

Purified peptidoglycan sacculii" appear as a dense layer in thin

section, although they are thinner than the layer seen in whole cells

(16, 29).








The space between the rigid layer and the cytoplasmic membrane

is generally not stained, but is apparently not "empty" since the two

layers are never in contact. In Nitrosocystis oceanus globular material

was observed in this area (72).

There is also a space between the rigid layer and the outer mem-

brane. In some organisms the material in this space is stained and

the rigid layer seems to be associated with the outer layer (27).

dePetris (15, 16) showed that this material sometimes appears globular.

Digesting the cells with proteolytic enzymes eliminates this material

and allows the outer membrane to separate from the rigid layer.

Digestion of isolated E. coli cell envelopes with trypsin, a procedure

which cleaves the lipoprotein from the peptidoglycan, also caused the

envelope to separate, but in this case the rigid layer was not visible

in the thin sections (10).

The outer double-track layer is identical in appearance to typical

membranes and is believed to be a lipid-protein bilayer. The LPS and

protein are probably arranged in a mosaic fashion, with the lipid

portion of the LPS extending into a phospholipid bilayer. Extraction

of cells with hot phenol-water removes the LPS and such cells are

devoid of the outer membrane (5, 16). With a marine pseudomonad it is

possible to remove the outer layer by washing the cells (22, 23).

This procedure yields isolated membrane material which contains most

of the LPS of the cells.

The 0-antigenic side chains of the LPS extend out from the outer

layer and are not stained in routine procedures. In thin sections of

packed cells there is generally a clear space between the outer layers

of adjacent cells, but with rough strains the outer membranes appear

to fuse.







Shands (61) used ferritin-labelled antibody to directly demonstrate

the presence of the LPS in the outer membrane of E. coli and Salmonella.

He observed that the ferritin molecules extended for considerable

distances from the outer double-track and concluded that there was a

large amount of unstained polysaccharide on the cell surface. Similar

results were obtained with Veillonella (37). It is not known if the

LPS is also located on the inner surface of the outer membrane, but the

apparent area required for the side chains would make such an arrange-

ment unlikely.

Freeze-etching studies of gram-negative bacteria.--In recent

years the new technique of freeze-etching has been used to study the

structure of cells (39, 40). In this process a sample is flash frozen,

placed in a vacuum chamber, and fractured. Ice is sublimed (etched)

from the fracture surface, and the surface is shadowed with evaporated

metal and replicated with evaporated carbon. The replica is cleaned

of adhering cellular material and is ready for examination in the

electron microscope. This procedure reveals the surface topography

of the fracture faces and the faces exposed by etching. A modification

of this method, double-recovery or complementary replica technique,

allows the replication of both surfaces produced when the specimen

is fractured (42, 67). By observing the same cell in both replicas,

it is possible to directly demonstrate the association of the various

fracture faces.

One aspect of freeze-etching that caused considerable confusion

and controversy for a period of years is the concept of membrane

splitting. When membranes are freeze-etched, two surfaces, one concave

and one convex, are produced. These surfaces were originally thought








to be the inner and outer surfaces of the membrane. Branton (6)

proposed an alternate interpretation, suggesting that membranes split

along an internal hydrophobic plane revealing two complementary surfaces

which do not represent the true surfaces of the membrane. Pinto da

Silva and Branton (49) proved this by labelling the outer surface of

red blood cells with ferritin and showing that the fracture face was

unlabelled and the labelled surface was only exposed by etching.

These results were verified by double-replica technique, by which it

was demonstrated that the convex and concave faces are produced by the

fracture of a single membrane in a particular cell (41).

The structure of the cell envelope of gram-negative bacteria has

been examined by freeze-etching (50). In both gram-positive and gram-

negative bacteria the cytoplasmic membrane fractures,producing particle

covered convex and concave faces (45, 65, 71). The convex face is

studded with a large number of particles, often arranged in a netlike

array. The concave face is sparsely covered with particles and some-

times appears pitted. Between the particles the surface is smooth,

and occasionally particle-free areas are present (44, 65). Fill and

Branton (19) observed that in magnesium starved E. coli the particles

are arranged in regular arrays and there are large particle-free areas.

The nature of these particles, which are also seen in a variety of

other freeze-etched membranes, has not been clearly established, but

they probably represent proteins which are intercalated into the lipid

bilayer (7, 64). Membranes which have little or no protein, such as

myelin sheath or artificial lipid bilayers, do not have particles on

their fracture faces (7).

The cell envelope of gram-negative bacteria fractures in at least








one other plane. Nanninga (44) observed that the cell wall of glycerol-

treated E. coli fractures revealing a rough irregular convex face and

a concave face which is composed of tightly packed flattened subunits

approximately 10 nm in diameter. He suggested that the subunits rep-

resented a protein layer superimposed on the peptidoglycan. In a later

study Van Gool and Nanninga (71) prepared complementary replicas and

proved that the two faces are apposed. By this time the idea of mem-

brane splitting had been established, and they concluded that this

fracture split the outer membrane in a manner analogous to the splitting

of the cytoplasmic membrane. This interpretation requires that the

subunit layer be superimposed on the inner surface of the outer half

of the outer membrane.

Gilleland et al. (26) demonstrated a similar fracture plane in

glycerol-treated P. aeruginosa, and they also concluded that it was

located in the outer membrane. The concave face in P. aeruginosa

appears as a smooth surface partially covered with spherical units

which are somewhat smaller than the subunits seen in E. coli.

Extraction of the cells with EDTA solubilizes proteins and yields

osmotically fragile cells. This treatment eliminates the spheres on

the concave fracture face. The protein can be restored to the cell

wall reversing the effect. P. aeruginosa was also studied by Lickfeld

et al. (32). A similar fracture was observed and was again postulated

to be a fracture of the outer membrane.

In the E. coli (71) and Pseudomonas (26) studies the cells were

suspended in glycerol as a cryoprotective agent. When these cells

were freeze-etched without glycerol the convex fracture face was

smooth. Both groups suggested that the smooth surface was the same









as the rough convex face and its appearance was altered by glycerol.

Neither study mentioned a concave fracture face in cells without

glycerol. Similar results were observed in Nitrosomonas europaea (70).

DeVoe et al. (17) observed a smooth convex fracture face in a

marine pseudomonad freeze-etched without glycerol, This surface was

also seen in spheroplasts produced by lysozyme digestion. No cell wall

fracture plane was present in cells which had been treated to remove

the outer membrane.

Nitrosomonas oceanus, an organism with a complex cell wall, was

examined by Watson and Remsen (72). They freeze-etched the bacteria in

a salt solution without glycerol and observed a concave globular layer

similar to the one seen in E. coli. According to their interpretation,

the corresponding convex layer is another globular layer obscured by a

thin rough layer. They concluded that the cell wall split at the

level of the peptidoglycan and the globular concave layer separated

the peptidoglycan from the outer membrane.

The outer surface of the cell envelope has been demonstrated by

etching of preparations without glycerol. The surface is often

smooth, but a fine subunit structure has been reported in E. coli and

P. aeruginosa (4, 19, 26). Large subunit structures have been observed

on the surfaces of a few bacteria, but these are clearly extra layers

external to the outer membrane (50).

In this study it will be shown that the cell envelope of the marine

vibrio MW40 is typical of gram-negative bacteria. When freeze-etched

under a variety of conditions the cell envelope fractures in three

planes. The location of the fracture planes and the nature of the

fracture faces are discussed and a model is presented which is consistent





12


with the current understanding of the freeze-etch process and the

structure of the cell envelope as determined by other techniques.















MATERIALS AND METHODS


Organism and maintenance of cultures.--The organism used in this

study was isolated in this laboratory from a sample of water from the

Waccasassa River estuary on the west coast of Florida and was designated

marine vibrio MW40. It is a marine organism requiring added salts for

growth in ordinary culture media and lysing in distilled water. It is

one of nine strains studied phenotypically in a preliminary study

(unpublished results). It was found that the organism is a gram-negative

polarly flagellated slightly curved rod, which is oxidase positive,

fermentative on glucose, sensitive to the pteridine 0/129, and has a GC

ratio of approximately 43%. It produces an insoluble blue-black extra-

cellular pigment which is probably indigoidine. Using the scheme of

Shewan (63) the organism would be classified as a Vibrio. The organism

has sheathed flagella and would be classified by Baumann as a member of

the genus Beneckea (1). It appears to be similar to the organism he

named Beneckea nigrapulchrituda (2).

Stock cultures were maintained on plates of a medium containing

0.5% gelatin (Difco Laboratories, Detroit, Mich.) and 2% agar, in a

salt solution, hereafter designated "complete salts," consisting of

0.22M NaCI, 0.026M MgCl2, and 0.01M KCI.

Culture and harvest of cells.--Cells were cultured in a medium

containing 0.5% trypticase (Baltimore Biological Laboratories, Baltimore,

Md.) and 0.1% yeast extract (Difco) in complete salts. The trypticase








and yeast extract medium was prepared at twice the final concentration

and adjusted to pH 7.6 with KOH before mixing with an equal volume of

double strength complete salts.

For routine work cells were cultured in 50 ml of medium in a 250 ml

flask, or in 250 ml of medium in a 1000 ml flask, incubated on a rotary

shaker overnight at 25 C. Cells were harvested by centrifugation at

3000 x g for 10 min. Except where otherwise noted, all centrifugation

was perfomred at 1-4 C in a Sorvall RC-2B refrigerated centrifuge using

an SS-34 rotor (Ivan Sorvall, Inc., Norwalk, Conn.).

Large quantities of cells were cultured in 11 1 of medium in a

20 1 carboy incubated at 25 C with aeration until late exponential

phase of growth, giving an approximate cell concentration of 1.5 x 109

cells/ml. The culture was cooled to 0 C by addition of 4 1 of ice and

sufficient NaCI (50 g) to maintain the salt concentration. Cells were

harvested in a precooled Delaval cream separator (Delaval Separator Co.,

Poughkeepsie, N. Y.).

Preparation of peptidoglycan.--Cells from an 11 1 batch culture

were suspended to 200 ml in complete salts at 0 C. The suspension was

rapidly heated to 60 C by adding 1 1 of complete salts preheated to

70 C, and placing in a water bath at 60 C for 10 min. The cells were

lysed by addition of 200 ml of a 28% (w/v) solution of Triton X-100.

The mixture was stirred for 10 min and then transferred to an ice-water

bath and cooled to about 4 C. When the temperature had fallen to 37 C,

500 ug of deoxyribonuclease (DNase) were added to reduce the viscosity.

The suspension was centrifuged at 10,000 x g for 90 min in a GSA rotor.

The pellets were washed once in 1200 ml of 0.1M NaCl and divided

into two equal portions. One half of the material was resuspended to








200 ml in 0.01M tris (hydroxymethyl)-amino methane (Tris) buffer,

pH 8.2, containing 40 mg of trypsin, and shaken for 30 min at 25 C.

A 120 ml volume of a solution of sodium dodecyl sulfate (SDS) was added

to give a final concentration of 4%. The mixture was shaken for 30 min

at 25 C, and then centrifuged at 27,000 x g for 1 hr at 20 C. The other

half of the material was suspended to 200 ml in 0.01M Tris buffer,

pH 7.2, without trypsin, mixed with SDS, and centrifuged as described.

The subsequent treatment was the same for both pellets.

The pellet was suspended in 50 ml of 4% SDS and added slowly with

stirring to 250 ml of boiling 4% SDS. After boiling for 5 min the

suspension was cooled slightly, shaken for 2 hr, and left overnight at

room temperature. The peptidoglycan was pelleted by centrifugation at

27,000 x g for 1 hr at 20 C, and washed once in 0.02M NaHCO It was

dialyzed for 48 hr against three changes of 0.02M NaHCO and two

changes of distilled water, and then lyophilized.

Peptidoglycan was also prepared by lysing cells in hot SDS. A

600 ml volume of culture was removed from the shaker, poured into 600 ml

of boiling 4% SDS, and placed in a water bath at 60 C for 30 min. The

suspension was cooled to 20 C and centrifuged at 16,000 x g for 1 hr

in a GSA rotor. The pellet was treated with boiling SDS, dialyzed, and

lyophilized as previously described. This procedure produced only a

small quantity of material, but it appeared to be whiter and purer than

the peptidoglycan prepared from the batch cultures.

Preparation of LPS.--LPS was extracted by a modification of the

hot phenol-water procedure (76). As suggested by O'Leary et al. (46)

a solution of MgCl2 was substituted for distilled water in this procedure.

Cells from a 11 1 batch culture were washed once in complete salts and





16

resuspended in 200 ml of 0.05M MgCl2. The suspension was mixed with an

equal volume of 90% phenol held in a water bath at 60 C, and the mixture

was shaken vigorously for 30 min. The mixture was cooled to 20 C and

certrifuged at 1500 x g for 30 min. The top aqueous phase containing

the LPS was removed and saved, and the phenol phase and the interface

were reextracted with an equal volume of fresh 0.05M MgCl2.

The aqueous phases were combined and centrifuged at 27,000 x g

to remove debris. The supernatant was dialyzed for 36 hr against

several changes of 0.01M MgCl2 and then lyophilized.

The crude LPS was purified by a modification of the method of

Romeo et al. (52). The LPS, 2 mg of ribonuclease (RNase), and 50 ug

of DNase were suspended in 60 ml of a solution consisting of 0.01M

Trisacetate buffer, pH 7.5, 0.1M NaCl, 0.01M MgCI2, and 0.01M sodium

azide. The suspension was incubated with stirring for 24 hr at 37 C,

and then dialyzed for 4 hr against 3 1 of the same salt solution minus

the NaCI. A 1 mg quantity of protease was added to the suspension in

the dialysis bag, and it was incubated for 24 hr at 37 C. The solution

was diluted to 100 ml with 0.01M MgCl2 and centrifuged at 96,000 x g

for 8 hr in a Beckman Model L-2 ultracentrifuge using a Ti50 rotor

(Beckman Instruments, Inc., Palo Alto, Calif.). The pellets were

resuspended to 40 ml in 0.01M MgCl2, 160 ml of 95% ethanol vere added,

and the mixture was stored overnight at 4 C to allow complete precipita-

tion of the LPS. The LPS was collected by centrifugation at 10,000 x g

for 30 min in a GSA rotor, resuspended in double-distilled water, and

lyophilized.

Preparation of cell envelopes using the French pressure cells.--

Except where otherwise noted, all of the manipulations involved in








preparing cell envelopes, cell walls, and isolated outer membranes were

performed at 0-4 C, and the final products were stored at 0 C until

processed for electron microscopy.

Cells from a 250 ml culture were suspended in 35 ml of 0.05M MgCI2

containing 50 ug of RNase and 50 ug of DNase. The cells were lysed by

passing the suspension thru a French pressure cell (American Instrument

Co., Silver Springs, Md.) operated at a pressure of approximately

16,000 psi. The lysate was diluted with an equal volume of distilled

water and centrifuged at 17,000 x g for 1 hr. The pellet was re-

suspended in 0.01M MgCl2 and centrifuged at 3,000 x g for 15 min to remove

whole cells. The supernatant was decanted and centrifuged at 17,000 x g

for 1 hr to pellet the cell envelopes.

Preparation of cell walls using Triton X-100.--Cells from a 250 ml

culture were resuspended in 250 ml of complete salts containing 50 ug

of RNase and 50 ug of DNase. The cells were lysed by the addition of

5 ml of a 50% (w/v) solution of Triton X-100 and the mixture was shaken

for 10 min. The cell walls were pelleted by centrifuging at 17,000 x

g for I hr, and washed once in a solution containing 0.05M MgC12 and

0.01M Tris buffer, pH 7.5. The pellet was resuspended in 40 ml of the

Tris-Mg solution and divided into two portions. A 0.5 mg quantity of

lysozyme was added to one of the suspensions and both were incubated

for 5 min at 37 C in a water bath. The suspensions were cooled and

centrifuged at 17,000 x g for 1 hr.

Preparation of crude outer membranes.--Crude outer membranes were

released from the cells by a modification of the procedures of Forsberg

et al. (22). Cells from a 250 ml culture were washed once with complete

salts and three times with 0.5M NaCl by resuspending them in 200 ml







volumes of the wash solution and centrifuging at 3000 x g for 10 min.

The final pellet was resuspended in 100 ml of 0.5M sucrose and placed

in a rotary shaker for 30 min at 20 C. The cells were pelleted by

centrifuging at 7500 x g for 15 min. The supernatant was carefully

removed and recentrifuged to remove any remaining whole cells. MgCl2

was added to the suspension to give a final concentration of 0.01M,

and the cell wall material was pelleted by centrifuging at 96,000 x g

for 2 hr. The pellets were resuspended, pooled in one tube, and

centrifuged as before.

Preparation of anti-LPS antiserum.--Three antigen preparations

were used: (1) whole cells treated briefly in a blender to remove

flagella and suspended in complete salts; (ii) cell walls prepared in

Triton X-100 and suspended at a concentration of 1 mg/ml in 0.9%

NaCI; (iii) partially purified peptidoglycan prepared by treating

cell walls with warm SDS and suspended at a concentration of I mg/ml

in 0.9% NaCl.

New Zealand white rabbits were injected subcutaneously with I ml

of antigen. Four injections were given at two-week intervals, and

the animals were bled two weeks after the final injection. Serum

was hated at 56 C for 30 min to inactivate complement, sterilized

by Millipore filtration, and frozen.

The activity of the antisera was determined by the agglutination

of bacterial cells. Cells were washed and resuspended in complete salts

solution to a concentration of approximately 5 x 108 cells/ml. 0.2 ml

of this suspension was mixed with 0.2 ml of serial dilutions of serum

and incubated at 30 C for 30 min. The degree of agglutination was

determined by microscopic examination.








Antiserum was adsorbed with LPS by mixing the serum with an equal

volume of complete salts containing purified LPS at a concentration

of 100 ug/ml. The mixture was incubated for 2 hr at 30 C and then

centrifuged at 27,000 x g for 15 min. The supernatant was carefully

removed and centrifuged again.

Labelling of cells with ferritin-conjugated antibody.--The in-

direct method was used to label the LPS on the cell surface with

ferritin-conjugated antibody. Cells were suspended to a concentration

of approximately 9 x 107 cells/ml in a dilute medium containing 0.1%

trypticase in complete salts. A 30 ml volume of this suspension was

mixed with 5 ml of rabbit anti-LPS antiserum and incubated for 30 min

at 30 C. The cells were pelleted by centrifugation at 12,000 x g for

10 min, washed three times with 40 ml volumes of trypticase medium,

and resuspended in 10 ml of the same medium. To this was added 0.5 ml

of commercially prepared ferritin-conjugated IgG fraction of goat anti-

rabbit immunoglobulin (IgA + IgG + IgM) (Cappel Laboratories, Inc.,

Downingtown, Pa.). The suspension was incubated for 30 min at 30 C,

diluted to 40 ml with trypticase medium, and centrifuged at 3000 x g

for 10 min. The pellet was washed once in 40 ml of complete salts,

and resuspended in 20 ml of complete salts. The suspension was divided

between two 15 ml glass centrifuge tubes and centrifuged at 3000 x g

for 10 min, One pellet was fixed for thin sectioning and the other was

resuspended in 10 ml of 0.05M MgCI2 and repelleted. It was then re-

suspended in several drops of the supernatant and transferred to a

plastic microtube. This tube was placed in a large tube and centrifuged

at low speed for several minutes. The microtube was cut off just above

the pellet and the cells were transferred to specimen holders for freeze-







etching. This procedure was necessary because of the very small size

of the pellet.

In some experiments, cells were fixed in glutaraldehyde before

antibody labelling. Cells from 40 ml of culture were fixed for 1 hr

in 20 ml of 4% glutaraldehyde in buffered salts (see section on thin

sectioning). A 20 ml volume of 0.5% trypticase medium was added and

the suspension was centrifuged at 3000 x g for 10 min, The cells were

resuspended in 40 ml of dilute trypticase medium and incubated for 1 hr.

This treatment was designed to eliminate any unreacted glutaraldehyde

and prevent non-specific uptake of antibody. The cells were pelleted,

resuspended in dilute trypticase medium, and labelled with antibody

as previously described.

Except for the incubations with antisera, all manipulations were

carried out at 0-4 C.

Thin sectioning.--Cells were fixed for thin sectioning in 4%

glutaraldehyde in a buffered salts solution consisting of 0.22 M tlaCl,

0.01M MgCl2, 0.01M KCL, and 0.02M potassium phosphate buffer, pH 7.4.

The concentration of Mg ion was reduced from the concentration in

complete salts to prevent the formation of a precipitate with the

phosphate buffer.

A 4 ml volume of the 4% glutaraldehyde fixative was added to 40 ml

of an actively growing culture and the suspension was set at 4 C for

30 min. Cells were harvested by centrifugation resuspended in 10 ml

of fresh fixative, and set for 2 hr in the cold. Cells were pelleted,

washed three times in buffered salts, and pelleted again. The super-

natant was carefully removed and 2 ml of 2% OsO4 in buffered salts was

added without resuspending the pellet. The cells were fixed in the








cold for 2 hr, and then washed with repeated changes of buffered salts

and finally with distilled water. The cells were repelleted as

necessary.

Cells were dehydrated thru a series of 25, 50, 75, 95, and 100%

ethanol and placed in acetone for embedding in plastic. The pellet was

broken into small pieces and embedded in an Epon-Araldite mixture by

the method of Mollenhauer (38).

Antibody labelled cells were fixed and embedded as described,

omitting the initial glutaraldehyde fixation in the medium. Cells

which were fixed with glutaraldehyde prior to antibody labelling were

postfixed with OsO4 and embedded as described.

Isolated cell envelopes were fixed by resuspending in 4% glutaralde-

hyde in phosphate buffered 0.01M MgCl2, postfixed in Os04, and embedded

as described for whole cells.

Embedded material was sectioned with a diamond knife (DuPont de

Nemours and Co.) in a Porter-Blum MT-2 ultramicrotome (Sorvall, Inc.,

Norwalk, Conn.). Sections were poststained with lead citrate (51) and

uranyl acetate.

Positive and negative stains.--Isolated cell envelopes were

positively stained by mixing a suspension with 0.5% aqueous uranyl

acetate. A small drop of the mixture was applied to a formvar coated

grid and immediately drawn off with filter paper. Purified LPS was

suspended in 0.01M MgCl2 and stained with uranyl acetate as described.

Purified peptidoglycan was suspended in distilled water and negatively

stained with potassium phosphotungstate. Grids were examined in the

electron microscope immediately.

Freeze-etching.--Glycerol-treated cells were prepared by mixing a








culture with an equal volume of 40% (v/v) glycerol in complete salts

and incubating for 15 min at 4 C. Other specimens were frozen in the

appropriate suspending medium without cryoprotective agents. Pellets

of the various materials were resuspended in very small quantities of

the supernatant and transferred to specimen discs with a drawnout

Pasteur pipette. Gold specimen discs with a central hole were routinely

used. For very small specimens, such as antibody labelled cells, gold

discs without a well were used. Specimens were frozen by plunging

them into liquid Freon 22 held at -150 C or a mixture of liquid and

solid N2 at a temperature of -209 C.

Specimens were freeze-etched according to the methods of Moor and

Muhlethaler (40) using a Balzers BA 360M freeze-etch apparatus (Balzers

High Vacuum Corp., Santa Ana, Calif.). The specimen was transferred to

the precooled (-150 C) stage and placed under a high vacuum. The

specimen temperature was raised to -100 C and it was fractured with a

cold microtome until a smooth surface was obtained. The surface was

etched for 0 to 2 min and then replicated.

The replicas were cleaned by floating them on 50% (v/v) Chlorox

overnight and rinsed on several changes of distilled water. Replicas

of ferritin labelled cells required an additional cleaning on 40% (w/v)

chromic acid. Replicas were picked up on uncoated 300 or 400 mesh grids.

Double-replica freeze-etching.--Complementary replicas were prepared

by the methods of Muhlethaler et al. (42). A suspension of glycerol-

treated cells was frozen in a sandwich of grids (28), Two 3 mm copper

discs were flared using a special press. Two nickel London finder grids

were similarly bent. One of the finder grids was placed on a copper

disc and a small quantity of vaseline was applied to the edges. The









second finder grid was placed on top of this and carefully adjusted so

that the corresponding grid squares were superimposed. The cell sus-

pension was applied to the grids, being sure that it penetrated through

the grid openings of both grids. The second copper disc was set on

top and the sandwich was clamped with a pair of forceps. The excess

liquid was removed with filter paper and the sandwich was plunged into

liquid-solid N2.

The sample was fractured in the Balzers apparatus using a special

hinged holder (custom designed and manufactured in this laboratory accord-

ing to the design of Muhlethaler et al. (42)). The holder replaces the

usual specimen stage. After fracturing, the specimens were replicated

immediately without etching. The finder grids with the attached

replicas were carefully separated from the copper discs and allowed to

dry. The grids were placed in chloroform to remove the vaseline and

then removed and dried. Replicas were cleaned overnight in 40% chromic

acid. Distilled water was added to dilute the acid and the grids were

transferred to fresh water. They were then removed and dried. If

the manipulations were done carefully the replicas remained attached

to the finder grids. This facilitated finding the corresponding halves

of cells in the two replicas.

Electron microscopy.--All materials were examined with a Hitachi

HUII-C or HUII-E electron microscope (Hitachi, Ltd., Tokyo, Japan)

operated at an accelerating voltage of 75 Kv. Photographs were taken

on DuPond Cronar film.

Amino acid anlaysis.--Amino acids and amino sugars were determined

on a JEOL model JLC-5AH automated amino acid analyzer (JEOL U.S.A., Inc.,

Cranford, N. J.) according to Spackman et al. (66). Samples were

hydrolyzed in 4NHCI for 11 hr at 105 C in sealed ampules.





24


Reagents.--The following chemicals were used: glutaraldehyde, 8%

under N2 gas (Polysciences, Warrington, Penn.); Os4 (Engelhard

Industries, Newark, N. J.); and Triton X-100 (Sigma Chemical Co.,

St. Louis, Mo.). The following enzymes were obtained from Sigma:

ribonuclease, pancreatic, Type I-A; deoxyribonuclease, pancreatic:

lysozyme, eggwhite, Grade I; protease, fungal, Type V; trypsin,

pancreatic, Type III. All other reagents were reagent grade.














RESULTS


Thin sections of whole cells.--In longitudinal sections the vibrios

appeared as slightly curved rods with a fine structure typical of gram-

negative bacteria (Fig. 1). Two double-track layers, the cytoplasmic

membrane and the outer membrane, were visible in the cell envelope

(Figs. 1, 2, and 3). The outer membrane was wavy, but in places appeared

to be closely associated with the cytoplasmic membrane (Fig. 2). The

fragmented appearance of the outer membrane was probably due to its

undulating contour, since it appeared as a double-track only when it

was perpendicular to the plane of the section.

In some cells there was a dense layer inside the cytoplasmic mem-

brane (Figs. 1 and 3). The nature of this layer is unknown. The area

between the inner and outer membranes was often fuzzy, but no organized

intermediate layer was visible. The dense layer seen in E. coli (16)

is seldom seen in marine bacteria, possibly because the peptidoglycan

is very thin (24).

Purified peptidoglycan.--The method used to prepare peptidoglycan

was similar to that of Braun and Rehn (10) and involved the treatment of

crude cell walls with hot SDS. Cells were heated to inactivate auto-

lytic enzymes and were lysed with Triton X-100 to minimize the fragmen-

tation of the peptidoglycan which occurs during lysis of cells by

mechanical means.

In order to determine if there was a lipoprotein covalently attached

to the peptidoglycan, two samples were prepared. One sample was digested

25



























Figs. 1, 2, and 3.


Thin sections of cells fixed in glutaraldehyde and
OsO Note the double layered structure of the
celt envelope.


Fig. I. Longitudinal and cross sections of cells.
(x 80,000).

Fig. 2. Cross section of a cell showing the double-track
appearance of the cytoplasmic and outer membranes
and close association of the two (arrows).
(x 212,000).

Fig. 3. Cross section showing an extra layer (arrow) inside
the cytoplasmic membrane. (x 167,000).






27



















CM
6













.. .
4-*






















-I
.. ,
M, -,' -. ,.







"zq~ ip r


&


L
iX~








with trypsin, the other was not. The results of the amino acid analysis

of these two samples were the same (Table 1). Both contained the usual

amino acids and amino sugars found in peptidoglycan from gram-negative

bacteria, that is, glucosamine, muramic acid, alanine, glutamic acid,

and diaminopimelic acid, in a ratio of approximately 1:1:2:1:1, Other

amino acids were present in very small amounts. Although the trypsin-

treated sample was slightly cleaner, the results indicated that there

was no lipoprotein attached to the peptidoglycan.

With the conditions used in the amino acid analysis neither muramic

acid and serine nor diaminopimelic acid and methionine could be

separated. Since the amounts of the other amino acids not normally

found in peptidoglycan were low, it was unlikely that this affected

the results significantly.

It was possible that an autolytic enzyme was cleaving the lipo-

protein, especially during the 30 to 45 minutes required to harvest the

cells. To minimize this possibility a small sample of peptidoglycan

was prepared by pouring an actively growing culture directly into hot

SDS. The amino acid content of this sample was the same as before, and

there was no evidence of a covalently linked lipoprotein (Table 1).

When negatively stained, the peptidoglycan appeared as cell shaped

fragments which were distinctly fibrous (Fig. 4). The peptidoglycan

was quite fragmented and did not resemble the finely granular sacculii"

obtained from other bacteria. The preparation shown was treated with

trypsin, but the untreated material had the same appearance. No

particles or granules were seen in either preparation.

Purified lipopolysaccharide.--The primary purpose of preparing LPS

was to obtain a purified antigen to test the specificity of antisera,


















C

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4-

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-

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-- U

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u

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-
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O
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O
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E
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0





O






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0 0 -










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-7
















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o N





-3 3- ~- N












.0 0 0
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CM r- i-. m-
CD Lu- .0 (V
-3- -3- -Q-

0 0 0 0


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o co

























C)
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C 0-







C) Cm





0 0














-)









C0 m0
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oa 2

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Fig. 4. Purified trypsin-treated peptidoglycan negatively stained with
potassium phosphotungstate. Note the fibrous nature of the
layer. (x 224,000).







































'I*


'* .


4."

4%'.


E-(r







but the general nature of the LPS was also of interest. The standard

method of preparing LPS from smooth strains of bacteria, phenol-water

partition, was used to obtain crude LPS, and it was further purified by

enzymatic treatments.

The results of preliminary work on the chemical analysis of the

LPS indicated that it was reasonably pure and similar to that found in

other bacteria. Keto-deoxyoctanoic acid and heptose were present (18,

75)- Amino acid analysis demonstrated three major components, probably

amino sugars, and small amounts of the various amino acids.

When uranyl acetate-stained preparations were viewed in the

electron microscope the LPS had the appearance of ribbons of discs

(Figs. 5 and 6). Similar results were reported by Shands et al. (62)

for LPS from Salmonella. Only a few areas appeared to have a double-

track structure.

Freeze-etc' ir of c. I. .' j'. c1 rol .--When cells were freeze-

fractured in a salts solution without glycerol the cell envelope

fractured in two planes, splitting the cytoplasmic membrane and the cell

wall (Figs. 7-14). The fracture faces of the cytoplasmic membrane were

similar to those seen in other bacteria. The convex face was a smooth

surface densely covered with particles, 4 to 8 nm in diameter, which

were sometimes arranged in a netlike array (Figs. 7-10). The concave

face was more sparsely studded with particles (Figs. 7, 12, 13, and 14).

In some preparations there were large circular areas devoid of particles

on both the convex and concave fracture faces (Figs. 7, 9, and 12).

There was no c F-rent correlation between the growth conditions of the

cells and the presence or absence of these particle-free areas.

Generally all or none of the replicas prepared from a single batch of



























Fig. 5. Purified LPS positively stained with uranyl acetate showing
ribbon structure and occasional areas (arrows) with double-track
appearance. (x 168,000).

Fig. 6. Purified LPS positively stained with uranyl acetate showing
ribbons and discs. (x 168,000).










/






5







cells demonstrated this feature, which indicates that it was a function

of the condition of the cells and not an artifact of the freeze-etching

process.

The cell envelope also fractured at another level, although with

much less frequency and generally only in small areas. This fracture

revealed two smooth faces and was probably splitting the outer layer.

The convex face was often seen as patches of cell wall material on the

cytoplasmic membrane (Figs. 7, 8 and 9). These patches appeared to

originate from the outer cell wall layer (Figs. 7 and 9). The corre-

sponding concave face was seen as holes in the cytoplasmic membrane

(Figs. 7, 12, and 13). The location of these patches did not appear

to be related to the particle-free areas on the cytoplasmic membrane.

The relationship between the smooth fracture face and the outer

cell wall layer was more clearly shown in etched preparations (Fig. 11).

Etching of preparations containing considerable amounts of salts often

revealed a eutectic layer surrounding the cells (Fig. 8, and ref. 17).

In some cases the eutectic layer was very thin, and although very fine

surface detail may be obscured, the general structure of the surface

was seen. This was demonstrated by the presence of flagella on the

cell surface (Figs. 10 and 11). It was observed that the smooth convex

fracture face is revealed by fracturing away a thin piece of the outer

layer (Fig. 11).

Another concave fracture face was occasionally observed in cells

fractured without glycerol. This surface was composed of globular

subunits (Fig. 14). The corresponding convex face was not demonstrated

in these preparations.

A more regular paracrystalline array of subunits was sometimes

































Fig. 7. Freeze-etched cells suspended in complete salts without glycerol.
This low magnification view shows concave and convex fracture
faces. (x 50,000).




37














k: I 4.i
-. t'" .. *'.2." -.,-
S,W?"-"""N w' a .
5, j% ,r :' "





w. r-


1."



.. ? .0., ..
cl It .." i "".
d.: CMm-' ., ; .' .'





I.- ,-"*,'t.

LI!
:,: "" ,

.

d ~ ~ ~ ~ ~ ...L~:; -z ~





























Fig. 8. Freeze-etched cell suspended in complete salts, showing the
particle-studded convex fracture face of the cytoplasmic
membrane and a patch of cell wall material. Etching has
revealed a eutectic layer which sometimes surrounds the cells.
(x 61,500).

Fig. 9. Unetched convex fracture of cells without glycerol. Note the
smooth fracture face of outer membrane and relation of this
fracture to the outer surface (edge) of the cell. Large
particle-free areas of the cytoplasmic membrane face are shown
(arrow). (x 82,000).













































I I

.
CM


... .. -.




.. 4 I.
..u-
. -,1







..y 9. .to ,
a *


f O.--.
0


I k -
c-- .-^





























Fig. 10. Deep etched cell in complete salts, showing the smooth or
slightly granular outer surface of the cell exposed by etching.
Note the structure of the fractured sheathed flagellum and
unidentified filaments. (x 83,200).

Fig. 11. Etched cell in complete salts showing the smooth convex
fracture face of the outer membrane and the true surface of
the cell. Note the thickness of the fracture edge (arrow).
(x 142,000).


















2..
-^ -S. .'- .
/ :."* ,. ,:-:.. ,.- 'l .?
#, / \ -.. *'. '
|^4 -' -.'^ .<: :..
'^ ". ^\*:^^ L
BL *^ ^,^^^"^^1'
:C'''rlci^^
LI:..,i "


b -,s


flagellum


T a *




























Fig. 12. Concave fracture
particle-studded
and holes in the
fracture face of
(arrow) are also


of a cell in complete salts showing the
fracture face of the cytoplasmic membrane,
surface which reveal the smooth concave
the outer membrane. Particle-free areas
shown. (x 80,000).


Fig. 13. Concave fracture of a cell clearly showing the smooth fracture
face of the outer membrane. (x 68,000).

Fig. 14. Concave fracture of a cell showing a surface with distinct
subunit structure. (x 116,500).












































N


-,^y^


~l~c .......








seen (Fig. 15). Although it is not apparent in this figure, this

array is located on the inner surface of the cytoplasmic membrane and

possibly corresponds to the extra layer seen in thin sections (Fig. 3).

Freeze-etchinq of alycerol-treated cells.--The primary fracture

plane in glycerol-treated cells was the cytoplasmic membrane, and the

fracture faces were indistinguishable from those seen in cells without

glycerol (Figs. 16 and 17). The outer layer also fractured revealing

the two smooth faces previously described (Figs. 18 and 19). Frozen

solutions of glycerol and water do not sublime significantly and there-

fore the outer surface of the cell could only be seen as an edge

(Fig. 19).

A third fracture commonly occurred which revealed a rough convex

surface and a concave surface composed of globular subunits (Figs. 20,

21, 22, and 23). This fracture plane has been demonstrated in other

bacteria and generally was thought to split the outer membrane (26, 32,

71). Our results suggest that a -ore plausible interpretation is that

the wall splits between the rigid layer and a globular protein layer

which separates the rigid layer from the outer membrane.

The convex surface of the rigid layer was generally seen only at

the ends of the fractured cell, with the envelope usually splitting at

the level of the cytoplasmic membrane (Fig. 20). The surface is rough

or granular with no clearly defined structure.

The concave globular layer appeared to be composed of globular

units approximately 10 nm in diameter (Figs. 22 and 23). In some areas

the subunits were in rows, but generally no ordered arrangement was

apparent. The layer was usually only partially visible, extending

from beneath the fractured cytoplasmic membrane. It was sometimes
































Fig. 15. Freeze-etched cell in complete salts showing a paracrystalline
array (arrow) inside the cytoplasmic membrane. a. (x 60,000)
b. (x 167,000).





46





.i. -. k




S ilS .. '

w a.;, . 'E .
**- '4 1.
....,
- -


^ t2' .1 $





U -..







i '.' ,4







-
-,1 -5b
'. t v



L -. iL*(
i< ^

2 -.^^ :


























Fig. 16.


Concave fracture of the cytoplasmic membrane in a glycerol-
treated cell. Cells were suspended in complete salts con-
taining 20% glycerol, The edge of the cell wall is visible
(arrow). (x 106,000).


Fig. 17. Convex fracture of the cytoplasmic membrane in a glycerol-
treated cell. Note the netlike arrangement of the membrane
particles. (x 107,000).







48














S1..6







* ...



" .- '6




























Unetched fractured glycerol-treated cell showing the concave
fracture face of the outer membrane. Note the width of the
shadow cast by the layer (arrow) indicating that it is quite
thin. (x 76,000).

Convex fracture of a glycerol-treated cell showing the smooth
fracture face of the outer membrane. In preparations with
glycerol the outer surface of the cell can only be seen as
an edge (arrow). (x 92,000).


Fig. 18.




Fig. 19.
















F: *

p..


1


p
y






























18


.'r .'.'
-p
* Ar


I -
aa
-e C
~




























Figs. 20 and 21.


Freeze-etched glycerol-treated cells showing the
rough convex fracture face of the rigid layer.


Fig. 20. (x 100,000).

Fig. 21. (x 157,500).












f.
r "


2~ ;r
4 t


4 I,.-
* *- 5 p '. * .
* a .' \ Y
* a "" ..
. v .

*
, ,, j ,,, ;'i. .

-* k ^ W ^
.. .r ,, t '.."- ,
r . ',- :


..,,.~ ;1.~~fc


" ". ," "'. "," "f ^ l s
S ,. a *.. . -L <
*) .;t^ hb -
-w r
--- a.f *. .- .

.,t,,: .
J'. -.' -CIS o. ... ,. ,. ,-^ & . -


























Fig. 22. Concave fracture of a glycerol-treated cell. Note the material,
apparently ice, on the surface of the globular layer.
(x 122,000).

Fig. 23. Concave fracture of the globular layer in a glycerol-treated
cell. a. (x 66,000), b. (x 167,000).

























//





54






e- -. --o. ,, .




t ..> .. '...




4L-







;! i- *^ ^^ '-: -' .,
^. '-" ?^ '.. '







*i% *. .. *w% ,,- . ... ..
.., '*. % .'
-\ "* '. ,








S...
23 3







obscured by an intermediate layer which appeared to be ice (Fig. 22).

This suggests that the fracture faces were separated by fluid prior

to freezing.

In areas where the globular layer was incomplete it was apparent

that the layer was in fact composed of individual subunits, and that

the subunits were backed by a smooth surface (Fig. 24). This smooth

surface was probably the true inner surface of the outer membrane as

opposed to the smooth fracture face of the outer membrane.

The cell envelope was also seen in profile in cross fractured

cells (Figs. 25 and 26). The inner and outer membranes were routinely

observed, and in some cases an intermediate layer was visible. The

membranes occasionally had a double-track appearance, but there is no

good explanation for this.

Using double-replica technique, complementary replicas of glycerol-

treated cells were prepared. The matching fracture faces of individual

cells were located in both replicas. In the figures shown, one of the

negatives was inverted before printing so that the images would appear

superimposable rather than as mirror images. The results of the double-

replica work clearly demonstrated that the three pairs of fracture

faces previously described were complementary, and each pair was

produced by a single fracture (Figs. 27, 28, and 29). It was also

apparent that while a rather thin layer was fractured away to expose

the smooth convex face, a thick piece of wall was removed in exposing

the rough convex face. This suggests that the rough face is at a

lower level in the cell envelope.

Structure of isolated cell envelope fractions.--The study of

isolated cell envelopes and various fractions of envelopes by freeze-

























Fig. 24. Concave fracture of a glycerol-treated cell. The globular
layer is incomplete and the individual subunits (arrow) and
the smooth backing layer are visible. The smooth surface is
thought to be the true inner surface of the outer membrane.
a. (x 60,000), b. (x 157,000).

Fig. 25. Cross fractured cell envelopes of three adjacent cells show-
ing the edges of the cytoplasmic membrane, rigid layer, and
outer membrane. The membranes have a double-track appearance.
(x 132,000).

Fig. 26. Edge fractured cell showing the fracture surface of the
membrane and the cross fractured cell wall. (x 132,000).



































IA 24a-






..-f., ."
")no
o w































Fig. 27. Complementary surfaces of a glycerol-treated cell observed by
double-replica technique. This pair of photographs clearly
shows that the membrane faces are produced by fracturing.
PHB, poly-B-hydroxybutyrate granule. (x 80,000).




59











; An., ,--,




r ; 7 ,1 .. > .t ii


7, .. ;z -


Or -W "
. .. -* '* -* ,. .J ..-

/27a'




;6 4<1




It Al"
~I~itr /fl.E / ~ RID'

~i Ii /I4k



I~ U27b



































Fig. 28. Complementary fracture faces of a glycerol-treated cell
showing the relationship between the concave globular layer
and the convex rough faces. (x 75,000).


























































a-


^V 1 ^" -* .? -A-'^ 1 1


f^ -
oo .

.- a** *- /' '^ ; ^ a
a- *- ,**..-
-..C.--, ,.' , .-.,
. -. r .. . , s . .-


28b


































Fig. 29. Complementary fracture faces of a glycerol-treated cell
showing the relationship between the smooth concave and convex
fracture faces. (x 100,000).































































I *, ,'p
iI t "9,'


-r ~ 3~
t.9, '








etching offers several theoretical advantages over using whole cells.

Since the fractions are of less complex composition, it should be

easier to correlate a particular fracture face with the cell wall

component which is being studied. In whole cells only those surfaces

which are natural fracture sites can be seen. Using isolated cell

envelopes it should be possible to observe other surfaces, such as

the inner surface of the cytoplasmic membrane, by merely etching away

the ice. In the case of marine bacteria there is an additional

advantage. Cell envelopes can be frozen in distilled water or dilute

salt solutions thereby reducing the tendency to form eutectic layers

which obscure etch surfaces.

Complete cell envelopes were prepared by lysing the bacteria in

a French pressure cell. Examination of thin sections revealed that a

variety of different structures were produced in this lysis process

(Figs. 30 and 31). The majority of the envelopes were double-membrane

structures which were either open C-shaped fragments or closed vesicles.

Single membrane vesicles were also seen and it was not possible to

determine if these were formed from the cytoplasmic membrane or the

outer membrane. As in whole cells, the rigid layer was not visible.

The various structures were also seen in uranyl acetate stained

envelopes (Fig. 32). Similar results were obtained with potassium

phosphotungstate and ammonium molybdate negative stains. No subunit

structure was observed in any of these preparations.

Freeze-etching of these complete envelopes yielded little

additional information. The cytoplasmic membrane fracture faces and

the globular layer were observed, but a variety of other surfaces

could not be identified (Figs. 33 to 36). Many of the envelope frag-




























Figs. 30 and 31. Thin sections of isolated cell envelopes prepared by
lysing bacteria in a French pressure cell. (x 127,000).

Fig. 32. Isolated cell envelopes positively stained with uranyl
acetate, showing double membrane fragments of various
sizes. (x 151,000).



































-4


32


li V -

























Figs. 33, 34, 35, and 36.


Freeze-etched isolated cell envelopes.
Envelopes were prepared by lysing bacteria
in a French pressure cell and suspended in
0.01M MgCl2.


Fig. 33. This fragment shows a typical cytoplasmic
membrane fracture face and the outer surface
exposed by etching. (x 116,000).

Fig. 34. An unfractured fragment exposed by etching.
(x 58,500).

Fig. 35. A concave fracture exposing the globular
layer. (x 58,500).

Fig. 36. This fragment shows the globular layer
apparently unfractured and exposed by
etching alone. (x 157,500).









k-*


A
\


4'


N
F' *


A '--...


L'


33I


A.


j







ments were unfractured and revealed by etching (Fig. 35). The enve-

lopes were suspended in 0.01M MgCl2 rather than in complete salts to

minimize the formation of eutectic layers. The true outer surface of

the envelopes appeared smooth or finely granular. The granularity

may represent the true structure of the surface or it may be an artifact

of very low angle shadowing since it is only seen in areas where the

surface is sloping away from the direction of shadow.

The true inner concave surface of the cytoplasmic membrane should

be revealed by etching but was not recognizable. Surprisingly, the

concave globular layer was exposed by etching (Fig. 36). This indicates

that this layer separated from the rough surface during the preparation

of the envelopes, and therefore could not be an internal fracture

surface of a membrane.

Cell walls were prepared by lysing cells with Triton X-100.

Schnaitman (59) has shown that in the presence of magnesium this deter-

gent solubilizes the cytoplasmic membrane leaving only slightly altered

cell walls. The freeze-etched appearance of these cell walls was very

complex and difficult to interpret. During preparation the walls appar-

ently packed together and flattened out, and possibly turned inside

out, making it difficult to recognize concave and convex surfaces.

A variety of surfaces were observed (Figs. 37 to 40). As expected,

no typical cytoplasmic membrane fracture faces were seen. Many of the

surfaces were composed of large circular structures which may have been

flattened vesicles (Fig. 38). Since the cytoplasmic membrane is absent,

the inner surface of the rigid layer should be exposed. This surface

was seen superimposed on the globular layer (Figs. 39 and 40).



























Figs. 37, 38, 39, and 40. Freeze-etched cell walls. The walls were
prepared by lysing cells with Triton X-100
and suspended in 0.01M IgC12. (x 90,000).

Fig. 37. This fragment has a smooth surface and shows
circular structures (arrows) of unknown nature.

Fig. 38. Cell walls showing large circular structures
which are probably flattened vesicles.


Figs. 39 and 40.


Fractured cell walls
layer and adjacent 1
not apparent whether
convex surfaces.


shown the globular
---s (arrows). It is
these are concave or










B~~E









When these walls were partially digested with lysozyme they had

a different appearance. Concave surfaces were observed which had a

distinct fibrous appearance (Figs. 41, 42, and 43). These surfaces

were exposed by etching. When this surface was fractured away the

globular layer was revealed (Figs. 42 and 43). This fibrous layer was

not seen in any other type of preparation and probably represents the

partially digested peptidoglycan.

Forsberg et al. developed a procedure which allows the removal of

the outer membrane from a marine pseudomonad (22, 23). Application of

this washing procedure to the marine vibrio caused the release of outer

membrane material, but did not markedly affect the viability of the

culture. The solubilized material was collected by ultracentrifugation,

and the pellet obtained was a clear gelatinous material unlike the

white, easily resuspended cell wall material produced in the other

procedures.

Freeze-etching of this material revealed variously shaped vesicles

which were seen in cross section and as concave and convex fracture

faces (Figs. 44 and 45). Slight etching exposed an edge around the

convex faces demonstrating that these faces are produced by fracturing.

Both of the fracture faces were smooth and appeared identical to the

smooth faces seen in whole cells. A few patches of globular layer were

also observed in these preparations (Fig. 44). These layers were

generally larger than the membrane vesicles and appeared flatter and

more cell shaped, suggesting that they were part of a larger fragment

of cell wall or had some innate structure which prevented them from

forming vesicles.
























Figs. 41, 42, and 43. Freeze-etched cell walls prepared by lysing cells
with Triton X-100 and partially digesting with
lysozyme.

Fig. 41. Note the fibrous nature of the concave surface
(arrows) and the patch of ice which indicates that
this is an etch surface. (x 66,000).

Fig. 42. This fracture shows the relationship of the
globular layer and the concave fibrous layer.
(x 66,000).

Fig. 43. In this area the fibrous material is emerging
from the ice background (arrow) indicating that
this is an etch surface. (x 100,000).





























. 3


944


,; l- ? .:


* G ." a 0
L b I .. ' ,I ,a

U . .
" "'


" '


-'.. Al
4 a




























Fig. 44. Freeze-etched isolated outer membrane material suspended in
0.01M MgCl2. Cross fractured vesicles and the smooth concave
and convex fracture faces are shown. Etching has revealed
the outer surface of a vesicle (arrow). A large fragment of
the globular layer is also shown. (x 86,000).

Fig. 45. Fractured outer membrane vesicles showing the fracture face
and unfractured surface (arrow). (x 135,000).









~ i~?: ,.


Ll44
s^
r^ I


P1


%
* i

V


45 *
I /l5.


,."


k 6 1


~m~t ~



Y








Localization of LPS with ferritin conjugated antibody.--Three

different antigen preparations were used to produce antisera. The

activities of the antisera were determined by agglutination of whole

cells. The highest titer was produced by injecting whole cells, but

it was found that adsorbing this antiserum with purified LPS would

reduce the agglutination activity by only 50O. Repeatedly adsorbing

with LPS had no further effect. Only the antiserum produced by in-

jecting partially purified cell walls was specific for LPS. This anti-

serum was of lower titer than the others, but its activity could be

completely adsorbed with LPS and it was used for all labelling experi-

ments.

Cells were labelled by the indirect method using anti-LPS anti-

serum followed by ferritin-conjugated anti-rabbit immunoglobulin anti-

serum. The specificity of the labelling was determined by treating

cells with anti-LPS antiserum or LPS adsorbed anti-LPS antiserum

followed by ferritin-conjugated antiserum. Cells were also treated

with ferritin-conjugated antiserum alone. The cells were examined in

the electron microscope without staining and the degree of labelling

was determined. The results are shown in Table 2. Neither unfixed nor

glutaraldehyde fixed cells were labelled by ferritin-conjugated anti-

serum alone, and they were only slightly labelled by LPS adsorbed anti-

serum. Using unadsorbed antiserum the fixed cells were more heavily

labelled than the unfixed cells. This may have been caused by loss of

labelled LPS from the cell surface during the washing procedure.

Thin sections of labelled cells revealed that the ferritin was

localized in a band external to the outer double-track layer (Figs.

46 and 47). In cells which were not fixed before labelling,the ferritin










Table 2. Determination of the specificity of antibody labelling.




Cells Treatment Result


Unfixed cells


Unfixed cells


Unfixed cells

Glutaraldehyde
fixed cells

Glutaraldehyde
fixed cells

Glutaraldehyde
fixed cells


Anti-LPS then ferritin-conjugated
antiserum

LPS adsorbed anti-LPS
then ferritin-conjugated antiserum

Ferritin-conjugated antiserum only

Anti-LPS then ferritin-conjugated
ant i serum

LPS adsorbed anti-LPS then
ferritin-conjugated antiserum

Ferritin-conjugated antiserum only


Labelled


Unlabelled


Unlabelled

Labelled


Unlabelled


Unlabelled























Figs. 46 and 47. Thin sectioned cells labelled with ferritin. The cells
were treated with rabbit anti-LPS antiserum, washed,
and then labelled with ferritin-conjugated goat anti-
rabbit antiserum,

Fig. 46. This cell was fixed following the antibody treatments.
(x 80,000).

Fig. 47. This cell was fixed in glutaraldehyde before antibody
treatments. Note the distance between the ferritin
molecules and the outer double-track. (x 167,000).


















9.








*, *,


r
I *,


---- CM


ferritin


46



I


' t

47

47


* b .%
* .* %
' t.*.


M* .. Lau


''








was limited to small areas. The labelling was heavier and more uniform

in fixed cells. In all cases the ferritin was a considerable distance

from the outer double-track. This is similar to the results of Shands

(61), and was probably due to the fact that the polysaccharide side

chains extend out from the cell surface and are not stained by the

heavy metal stain. The use of indirect labelling puts two antibody

molecules between the antigen and the ferritin molecules which could

account for a 20 to 50 nm space.

For freeze-etching, ferritin labelled cells were suspended in

0.05M MgCl2. It was found that this solution would maintain the

integrity of the cells and still allow the deep etching needed to

expose the cell surface. The presence of ferritin confirmed that the

outer surface of the cells was revealed by etching. It was difficult

to compare the appearance of the ferritin in thin sections and in

freeze-etching. All of the particles in freeze-etched cells were lying

close to the surface of the cell. It is possible that during the

freezing process the ferritin molecules are excluded from the growing

ice crystals thus packing them down onto the cell surface.

With unfixed cells the ferritin molecules were clustered in

patches on the cell surface (Figs. 48 to 51). The envelope fractured

normally revealing the convex cytoplasmic membrane and outer membrane

fracture faces (Figs. 50 and 51).

Glutaraldehyde fixed cells were heavily labelled (Fig. 52). The

ferritin was evenly distributed on the cell surface and many individual

ferritin molecules were seen. In unfixed cells the LPS was freely

mobile in the membrane and was probably drawn together by the divalent






























Figs. 48 and 49.


Freeze-etched unfixed cells labelled with ferritin and
suspended in 0.01M MgCl2. The LPS on the cell surface
was labelled indirectly with ferritin-conjugated anti-
body. The cell surface was exposed by deep etching.
The ferritin molecules are clustered in patches.
(x 92,000).




















S. "

i.... . . . .
IrzlptL jf .,.

































Figs. 50 and 51. Convex fractured unfixed cell labelled with ferritin.
Note the relationship of the smooth fracture face to
the ferritin labelled surface. (x 92,000).











































mrwY0


II


*'.4%


.. W4
^ '


~b~i~Ei~4r~i~i~sC~~;






























Fig. 52. Ferritin labelled glutaraldehyde fixed cell. The ferritin
molecules are more evenly distributed than in unfixed cells.
Note the relationship of the smooth convex fracture face and
the ferritin covered surface exposed by etching. The
fracture edge is quite thin (arrow). (x 157,000).





















S..' .








-r
ferritin.r ..--



'"- "P l


c.I' .
I I1n,


.


i/*'* ^


0 .
O -.


*- .- '-


1


.P 1
ar;S
..-I./.1
^





88


antibody. Glutaraldehyde fixation stabilized the membrane and pre-

vented the LPS from moving. The relationship between the smooth

fracture face and the ferritin labelled outer surface was clearly

shown by these experiments (Fig. 52).




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