Journal of General Microbiology (1978), 108,283-294. Printed in Great Brituin
283
An Electron Microscopic Study of the Location of Peptidoglycan in
Group A and C Streptococcal Cell Walls
By M. WAGNER A N D B. WAGNER
Research Centre for Molecular Biology and Medicine,
Central Institute of Microbiology and Experimental Therapy,
Academy of Sciences of the GDR, 69 Jena, German Democratic Republic
M. R g C
Department of Electron Microscopy, Institute of Hygiene and Epidemiology,
100 42 Prague, Czechoslovakia
AND
(Received 11 May 1978)
The morphological appearance of deproteinized Group A and C streptococcal walls after
treatment by different procedures extracting teichoic acids and polysaccharides (formamide,
hydrochloric acid, nitrous acid, trichloroacetic acid, sulphuric acid, sodium hydroxide and
sodium deoxycholate) was compared with the content of teichoic acids and polysaccharides
remaining in the treated walls. All procedures extracted teichoic acids almost completely,
but polysaccharides were extracted to various degrees. The ultrastructural appearance of
walls after these extractions still exhibited the triple-layered wall profile; only a reduction of
thickness of the wall and of electron density of the layers occurred. Therz was no direct
correlation between the reduction of rhamnose content and thickness of walls.
The ultrastructural localization of peptidoglycan in the streptococcal walls was explored
by means of the indirect immunoferritin technique using anti-peptidoglycan antibodies
isolated from anti-Group A-variant antisera. Ferritin particles were bound predominantly
to filamentous structures which protruded from both surfaces of peptidoglycan fragments
and isolated walls. Peptidoglycan was also detected on the filamentous protrusions of whole
cocci. These results contradict models of the streptococcal wall in which peptidoglycan
forms the innermost layer and support a mosaic structure in which peptidoglycan forms
a network of the peptidoglycan-polysaccharide complex.
INTRODUCTION
The chemical and morphological structures of the walls of Group A and C streptococci
are very similar (Krause & McCarty, 1962; Cole, 1968). Group A streptococcal wall
consists of four chemically defined components - proteins (M, T and R), polysaccharide,
peptidoglycan and teichoic acid (Swanson et al., 1969). In ultrathin sections three morphologically distinct layers can usually be distinguished (Cole, 1968; Swanson et al., 1969;
Wagner & Wagner, 1972~).The wall is composed of an electron-dense inner layer, a middle
layer of medium electron density and an outer layer which in most strains is covered by
filamentousprotrusions. The morphological appearance of the wall, and the fact that proteins
can easily be removed by proteolytic enzymes without affecting the viability of the streptococci (Lancefield, 1943), has led to the belief that all chemical components of the wall are
arranged in concentric layers. This concept is still held (Krause, 1972; Heymer et al.,
1973; Davis et al., 1973): thus, proteins form the filamentous protrusions, below which
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
M. WAGNER, B. WAGNER A N D M. R k C
284
polysaccharides are localized, with peptidoglycan as the innermost layer forming the
basal structure of the wall; the localization of teichoic acid is not considered.
One possible approach to localize individual components of the wall is to study the
appearance of walls after chemical extraction of different constituents. Group A streptococci can be deproteinized by proteolytic enzymes - partially by pepsin (Beachey & Ofek,
1976) and almost completely by trypsin (Swanson et al., 1969). The present investigation was
undertaken to establish the morphological and chemical features of streptococcal walls after
removal of polysaccharide and teichoic acid by different extraction procedures leaving more
or less pure peptidoglycan.
Immunoelectronmicroscopy (Singer, 1959; Nakane & Pierce, 1966; Faulk & Taylor, 1971)
allows a direct proof of the localization of antigenic cell components. In Group A streptococci, the M protein (Swanson et al., 1969; Wagner & Wagner, 1972a; Wagner et al., 1974)
and the group-specific polysaccharide (Swanson & Gotschlich, 1973) have both been
localized; and the use of the ferritin-labelledlectin from the edible snail Helix pomatia has
made it possible to localize the group-specific polysaccharide in Group C streptococci
(Wagner & Wagner, 1975). The antigenic nature of Group A and C streptococcal peptidoglycan has been known for a long time (Abdulla & Schwab, 1965; Karakawa & Krause,
1966). In the present study, peptidoglycan antibodies were used for the immunoelectron
microscopic demonstration of peptidoglycan in whole organisms and walls of Streptococcus
pyogenes and Streptococcus equisimilis. For comparison, antisera against Group A-variant
streptococci were used after absorption with HCl extracts from homologous strains.
METHODS
Bacterial strains and growth conditions. The following strains from the Collection of the Central Institute
for Microbiology and Experimental Therapy, Jena, were used: Streptococcus pyogenes (Group A) strains
N Y ~(type 12), ~ ~ (type
1 717) and Haskins (type 19); Streptococcus equisimilis (Group C) strain ~ 4 6 ~ .
Streptococcus pyogenes (Group A) strains 10/58 (type 6) and 63/50 (type 32) were from the Czechoslovak
National Collection of Type Cultures of the Institute of Hygiene and Epidemiology,Prague.
For the extraction studies, the bacteria were grown with shaking in Todd-Hewitt broth (Difco) for 4 h
at 37 "C and then treated twice with trypsin (Difco; 0.1 %, w/v, in 0.1 M-phosphate buffer, pH 7.8) at 37 "C
for 2 h. After treatment, the organisms were repeatedly washed in 0.1 M-phosphate buffer before being
subjected to the different extraction procedures.
For the immunoelectron microscopic studies, the bacteria were grown with shaking in Proteose broth at
37 "C overnight, then centrifuged and washed with R-K buffer (Ryter & Kellenberger, 1958).
The presence of the Fc-binding factor in these strains was determined by the method of Christensen et al.
(1976).
Preparation of isolated walls and peptidoglycan. Bacterial suspensions were shaken with glass ballotini in
a Braun disintegratoror a vibrating homogenizer (Centre for Scientific Engineering,Academy of Sciences of
the GDR). After differential centrifugation of the homogenate (Salton, 1964) and treatment with trypsin,
ribonuclease and deoxyribonuclease,successively, each at a concentration of 1 mg ml-l for 4 h at 37 "C,the
walls were finally washed three times in distilled water.
To prepare peptidoglycan, walls were extracted three times for 15 min with formamide at 160 "C and
washed with phosphate-bufferedsaline, pH 7-2 (PBS).
Extraction procedures and determination of rhamnose and phosphorus. Whole cells as well as isolated walls
were extracted by each of the following methods: (a) formamide, 160'"C, 30 min (Fuller, 1938); (b) HCl,
0-2M, 100 "C, 10 min (Lancefield, 1928); (c) mop,0.8 M, 37 "C, 15 min (Swanson & Gotschlich, 1973;
HN02was prepared from equimolar parts of NaN02and HCl); ( d )trichloroacetic acid (TCA), 10 % (v/v),
4 "C, 24 h (Schleifer & Kandler, 1967); (e) TCA, 10 % (v/v), 60 "C, 4 h (Schleifer & Kandler, 1967);
(f)H2S04,0.1 M, 60 "C, 24 h (Schleifer, 1975); ( g ) NaOH, 0.5 M, 60 "C, 4 h (Archibald et al., 1969);
(h) sodium deoxycholate, 1 % (w/v), 0 "C, 16 h (Hill, 1967). After extraction, the bacteria were recovered
by centrifugation and washed in 0.1 &phosphate buffer, pH 7.2, before being fixed for electron microscopical investigation. The extracted walls were recovered by centrifuging and the contents of rhamnose
(as a marker of polysaccharide)and phosphorus (as a marker of teichoic acid) were determined according to
Dische & Shettles (1948) and Chen et al. (1956), respectively.
Peptidoglycan antibodies. Since immunization of rabbits with isolated peptidoglycan did not result in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
Streptococcal pep t idog lycan localization
285
satisfactory antibody titres, antisera against whole organisms of Group A-variant streptococcus strains
T12 and ~ 5 were
0
used. As found by Karakawa et a/. (1968), rabbit antisera against Group A-variant
streptococci contained high titres of peptidoglycan antibodies. To isolate the antibodies, the peptidoglycan
fraction prepared from lyophilized Group A streptococcus (strain Haskins) walls (200 mg) was mixed with
antiserum (5 ml) and incubated at 37 "C with slight agitation for 1 h. The mixture was then centrifuged and
the sediment was washed once with PBS before being suspended in 0.1 M-glycine/HCl, pH 2.5 (2.5 ml).
After 5 min, the suspension was centrifuged and the supernatant containing the antibody was quickly
neutralized with 0.1 M-NaOH. Thorough washing with PBS rendered the peptidoglycan fraction fit for
absorption again. Pooled antibody solutions were concentrated against Aquacide (Calbiochem) to a protein
concentration of about 5 mg ml-l.
Absorption of Group A-variant streptococcal antisera. Antisera were absorbed three times with the HC1
extract (Lancefield & Perlmann, 1952) of the homologous Group A-variant strain. After this procedure, the
capillary precipitation reaction of absorbed antiserum with HCl extract was negative.
Ferritin-labelled anti-rabbit Ig G. Antiserum against rabbit IgG was prepared by immunizing a goat with
a mixture of increasing amounts of IgG (0-5 to 16 mg per injection) and Freund's complete adjuvant (Difco)
at 10 d intervals. The antigen was injected simultaneously at multiple sites. The precipitation titre of the
antiserum against 0.1 % (v/v) rabbit IgG was 1 : 128.
The IgG fraction was prepared from the antiserum by adding ammonium sulphate to half saturation
followed by chromatography of the precipitated globulin fraction on DEAE-cellulose(Levy & Sober, 1960).
The IgG fraction was conjugated with ferritin (five times crystallized, Research Institute for Vaccines,
Dessau, GDR) by means of glutaraldehyde (Wagner & Wagner, 1972b). The crude conjugate was freed of
non-labelled IgG and most of the non-labelled ferritin by chromatography on a column ( 2 3 x 100 cm) of
Biogel A 1.5 m (Calbiochem)eluted with PBS. Pooled purified fractions were concentrated against Aquacide
and sterilized by filtering (Millipore, 045 pm pore size).
Treatment with pronase and nitrous acid for immunoefectronmicroscopy. Whole organisms and peptidoglycan were incubated at 37 "C with shaking in sterile 0-02M-acetate buffer, pH 8.0,containing 0.1 % (w/v)
pronase (Serva) for 5 to 65 h. Extraction with HNO, was described above.
Electron microscopical techniques. For examination of extracted bacteria, glutaraldehyde (Serva) 2.5 %
(v/v) in 0.1 M-phosphate buffer, pH 7.2, was added to washed organisms. After 30 min fixation at 22 "C,
the organisms were centrifuged, washed overnight in 0.1 M-phosphate buffer, pH 7.2, centrifuged again, and
fixed in 1 % (w/v) OsOain R-K buffer containing 10 % (w/v) tryptone at 22 "Cfor 16 h. The fixed organisms
were embedded in 1 % (w/v) Noble agar (Difco). Small blocks with embedded cells were treated with uranyl
acetate for 90 min, dehydrated with acetone and embedded in Vestopal (Serva). Ultrathin sections prepdred
on a LKB Ultratome I were contrasted with lead citrate (Reynolds, 1963).
The thickness of walls was determined by micrometermeasurements of 25 to 40 organisms on photographs
of ultrathin sectionsat a final magnification af 80000 to 100000. Five different places on each organism including cell poles were chosen for measurement, the location of each being at some distance from the site of
septum formation where the thickness of the wall was usually greater. The distance between external surfaces
of the electron-dense layers on antitangentiallysectioned cells (a sharp appearance of both layers being the
indicator) was measured. Calibration of the electron microscope (EM300, Philips) with catalase crystals
showed less than 1.5 % error in magnification.
For labelling experiments, the indirect immunoferritin technique was used. Whole organisms, walls and
peptidoglycan were incubated with peptidoglycan antibody at 37 "C for 1 h with shaking, then washed
three times with R-K buffer and incubated with ferritin-labelled anti-rabbit IgG at 37 "C for 1 h. After
washing, the samples were fixed in 2.5 % (v/v) glutaraldehyde in 0.075 M-cacodylate buffer, pH 7.2, containing 0.17 M-sucrose and 0.05 % (w/v) CaCl, for 30 min at room temperature, washed five times with
the same buffer and embedded in 1 % Noble agar. Fixation in 1 % (w/v) OsO, in R-K buffer took place
overnight at 4 "C. After dehydration in acetone and contrasting with uranyl acetate and phosphotungstic
acid at the step of 70 % (v/v) acetone (Wohlfarth-Bottermann,1957), the samples were embedded in Epon.
Ultrathin sections were examined without poststaining. As controls, whole organisms, walls and peptidogIycan were incubated with ferritin-labelled anti-rabbit IgG alone. Also, glutaraldehyde-prefixedcells were
incubated with antibodies as above.
The electron microscopes used for examiningultrathin sectionswere an EM300 (Philips) and an Elmiskop I
(Siemens), both operating at an accelerating voltage of 80 kV, and a KEM (VEB Werk fur Fernsehelektronik, Berlin, GDR), operating at an accelerating voltage of 60 kV.
MIC
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
108
286
M. WAGNER, B. WAGNER A N D M. R 9 C
Table 1 . Rlhmnose content of walls of S. pyogenes (strain 10/58) and S. equisimilis
(strain ~ 4 6 after
~ ) various extraction procedures
S. pyogenes
S. equisimilis
h
Procedure
Control walls
(unextracted)
Formamide
HNO,
HCl
TCA (4 "C)
TCA (60 "C)
Hm,
NaOH
Sodium deoxycholate
*
r
Rhamnose
content *
Expressed
as % of
control
Rhamnose
content *
Expressed
as % of
control
15.80
100.0
23-00
100.0
0.90
6-14
3.86
13.50
4.65
11.50
1 1 -40
10.70
5.6
38.9
24.4
85.4
29.4
72.7
72.6
67.6
1 -02
7.50
4-65
19-65
5.45
18.52
15-30
18.50
4-4
32.6
20.2
85.4
23-7
80.5
66.7
80.5
Expressed as mg rhamnose (100 mg walls)-l.
All bar markers represent 0-2pm.
Fig. 1. Streptococcus pyogenes (strain 10/58) and S. equisimilis (strain ~ 4 6 ~untreated
),
and after
different extraction procedures. (a) S. pyogenes, untreated control. (b) S. pyogenes, treated with
trypsin. (c) S. equisimilis, treated with trypsin. ( d ) S. pyogenes, treated with hot formamide: the
thickness of the wall has been reduced although the triple-layered structure is preserved. ( e ) S. equisimilis, treated with hot formamide. (f) S. pyogenes, treated with hot formamide: in contrast to the
cocci shown in (a-e), the plasma membrane is detached from the wall.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
St reptococcal pept idoglycan localization
287
Table 2. Thickness of trypsin-treated walls of S. pyogenes (strain 10/58)
after various extraction procedures
Thickness of wall
A
I
Procedure
Control walls
(unextracted)
Formamide
HNO,
HC1
TCA (4 "C)
TCA (60"C)
J&SO,
NaOH
Sodium deoxycholate
Range
(nm)
18-24
Average
(nm)
20.5
10-13
12-18
12-18
16-20
12-15
12-18
10-15
16-20
11.9
14.6
15.5
17.9
13.5
15.0
12.5
17.8
\
Expressed
as % of
control
100.0
58.2
71-2
75.6
87.3
65.9
73.2
61.0
86-8
RESULTS
Extraction of polysaccharides and teichoic acids from walls
Polysaccharides were extracted from walls to varying degrees, formamide extraction
being the most efficient (Table 1). In contrast, all the procedures extracted essentially all
the teichoic acid, the residual walls containing only traces of phosphate.
Morphological features of walls after diferent extraction procedures
The ultrastructure of walls of the streptococcal Group A and C strains studied was
similar. Untreated walls (Fig. l a ) showed an inner electron-dense layer (5-5 to 8 - 5 nm),
a layer of medium electron density (10 to 15 nm) and an outer electron-dense layer (2.5 to
5 nm), bearing filamentous protrusions (20 to 40 nm) ;the number of protrusions was greatly
reduced by incubation with trypsin (Fig. l b , c). Table 2 summarizes the results obtained
for the thickness of Group A streptococcal walls after different extraction procedures.
Although the thickness of the wall was reduced to different degrees, the general morphological triple-layered appearance of the wall remained unchanged, both after the highly
efficient formamide extraction (Fig. 1 d to f) and after the other procedures used (Fig. 2a
to h). In no case was there complete loss of any layer. However, it was evident that with the
different modes of extraction used the reduction in wall thickness was accompanied by a
reduction in the electron density of the individual layers.
Table 3 compares the reduction in wall thickness of Group A streptococci after the
various extraction treatments with the degree of reduction of the polysaccharide content
of the walls. Generally there was no simple quantitative correlation between polysaccharide
content reduction and the thickness of wall remnants. This discrepancy was most obvious
after formamide treatment which reduced polysaccharide content by about 95 % but wall
thickness by only 40 %.
Localization of pept idoglycan
Incubation of peptidoglycan of S . pyogenes and S. equisimzlis with peptidoglycan antibody
followed by incubation with ferritin-labelled antirabbit IgG resulted in both sides of the
fragments becoming heavily labelled with ferritin (Fig. 3 a). On antitangentially sectioned
parts of the fragment, the ferritin particles were not attached directly to the visible peptidoglycan structure but seemed to be bound to short filamentous structures protruding from
both sides of the fragments. These filaments were not seen on non-incubated peptidoglycan,
which exhibited a tribanded profile in antitangential sections (Fig. 3 b). Incubation with
ferritin conjugate alone did not result in labelling of peptidoglycan.
19-2
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
288
M. WAG NE R, B. WAG N E R A N D M. Rq'C
All bar markers represent 0-2,urn.
Fig. 2. Streptococcus pyogenes (strain 10/58) and S. equisimilis (strain ~ 4 6 after
~ ) different extraction procedures. S. pyogenes: (a) after extraction with HNO,: note the irregularity in the thickness
of the wall residue. (b) After extraction with HCl: the triple-layered structure of the wall is preserved. (c) After extraction with TCA at 4 "C.(d)After extraction with TCA at 60 "C:as in (c) the
ultrastructure of the wall has been relatively well preserved, whereas the nucleoid and the cytoplasm have been destroyed. (e) After extraction with H,SO,: a pronounced decrease in the electron
density of individual parts of the wall has occurred. (f)After extraction with NaOH. (g) After
extraction with sodium deoxycholate. S. equisimilis : (h)after extraction with sodium deoxycholate.
Isolated walls of S. pyogenes and S. equisimilis bound peptidoglycan antibody on both
their inner and outer surfaces (Fig. 3 c). The pattern of labelling was the same as on peptidoglycan. Walls also showed labelling on filamentous structures which were not observed on
untreated walls (Fig. 3 4 .
Whole organisms also bound peptidoglycan antibody, the ferritin being found on filamentous protrusions of the outermost layer of the wall as well as immediately on the coccal
surface (Fig. 4a, b). The amount of attached ferritin particles differed from strain to strain.
In strains NYS, 10/58, ~ ~ and
1 Haskins,
7
incubation with ferritin-labelled anti-rabbit IgG
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
Streptococcal pep t idogly can -1ocalizat ion
289
Table 3. Comparison of the reduction in wall thickness with the reduction in polysaccharide
content of the walls of S. pyogenes (strain 10/58)
Procedure
Formamide
HNO,
HCI
TCA (4 "C)
TCA (60 "C)
HzS04
NaOH
Sodium deoxycholate
Reduction in
Reduction in
wall thickness rhamnose content
( % of control) (% of control)
41.8
28.8
24.4
12.7
34.1
26.8
39.0
13.2
94.4
61.1
75-6
14.6
70.6
27.3
27.4
32.4
All bar markers represent 0-2pm.
Fig. 3. Ferritin labelling of peptidoglycans and walls of S. pyogenes (strain NY5) and S. equisimilis
(strain ~ 4 6 ~(a)
) . Peptidoglycan from S. pyogenes, labelled with anti-peptidoglycan antibody
(indirect immunoferritin technique): ferritin particles cover filaments on both sides of the antitangentially sectioned fragment. (b) Peptidoglycan from S. equisimilis, unlabelled control : the
fragment shows a triple-layered structure with smooth surfaces. (c) Wall from 5'. pyogenes, treated
as in ( a ) :ferritin particles are situated on both sides of the wall where they seem to be located on
similar filaments to those observed on peptidoglycan. (d) Wall from S. equisimilis, unlabelled
control : the surface of the wall is smooth.
alone did not result in ferritin binding on the surface, whereas a few ferritin particles were
observed on the surface of strain ~ 4 6 ~ .
Incubation of whole organisms with Group A-variant antiserum followed by treatment
with the ferritin conjugate resulted in labelling of the surface in the same pattern as with
peptidoglycan antibodies (Fig. 5 a). Ferritin localization was also unchanged on organisms
pretreated with glutaraldehyde before incubation with antibody (Fig. 5b).
Extraction of cocci and walls with HNOz did not result in any changes of labelling pattern
with peptidoglycan antibody (Fig. 5 c). Similarly, after pretreatment with pronase for 5 to
65 h, cocci, walls and peptidoglycan were labelled with peptidoglycan antibodies. However,
the number of ferritin particles diminished after 65 h treatment and the structure of peptidoglycan seemed to be loosened. On both sides of the walls and peptidoglycan fragments,
filamentous substructures were observed which were partly labelled with ferritin (Fig. 5 d).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
290
M. WAGNER, B. WAGNER A N D M. R k C
All bar markers represent 0.2 pm.
Fig. 4. Streptococcus pyogenes, labelled with anti-peptidoglycan antibody (indirect immunoferritin technique). (a)Strain N Y ~ ferritin
:
particles cover the wall surface. (b) Strain ~ ~ 1 ferritin
7 :
particles cover only the tips of the filaments.
All bar markers represent 0.2 pm.
Fig. 5 . Streptococcus pyogerzes and S. equisimilis, labelled with anti-peptidoglycan antibody
(indirect immunoferritin technique). (a) S. pyogenes (strain 10/58), labelled with Group A-variant
streptococcal antiserum that had been absorbed with HCI extract: ferritin particles are present on
filamentous protrusions as well as on the wall surface. (6) S. equisimilis (strain ~ 4 6 ~ )
treated
,
with
glutaraldehyde before labelling: ferritin particles cover the wall surface. (c) S. pyogenes (strain N Y ~ )
treated with 0.8M-HNO, before labelling: the ferritin localization is comparable to that in untreated cells. ( d ) Wall from S. equisimilis (strain ~ 4 6 ~ )incubated
,
with pronase for 65 h before
labelling: the wall has filaments on both sides, some of which are covered by ferritin.
Injiuence of the Fc-binding factor’ on labelling
To study the influence of the Fc-binding factor (Christensen et al., 1976) on the specificity
of labelling, cells of strain 63/50 (which had a high content of Fc-binding factor) and strain
10/58 (which did not contain Fc-binding factor) were incubated with Group A-variant
antiserum and ferritin conjugate or with ferritin conjugate alone. Initially both strains were
heavily labelled with ferritin (Fig. 6a, c), but after incubation with the ferritin conjugate
alone, only strain 63/50 had ferritin particles present on the tips of filaments (Fig. 66, d).
DISCUSSION
The aim of this work was to localize peptidoglycan in the walls of Group A and C streptococci. If the streptococcal wall were composed from several layers identical with the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
Streptococcal pep t idoglycan localization
29 1
All bar markers represent 0.2 ,urn.
Fig. 6. Influence of the Fc-binding factor on the labelling of S. pyogenes. (a) Strain 10/58 (not
containing Fc-binding factor), labelled with Group A-variant streptococcal antiserum (indirect
immunoferritin technique) : the coccal surface is heavily labelled with ferritin. (b) The same strain,
treated with ferritin-labelled anti-rabbit IgG only, as a control: the surface is free of ferritin.
( c ) Strain 63/50 (having a high content of Fc-binding factor) treated as in (a): unspecific localization
of ferritin on the tips of filamentous protrusions. ( d ) The same strain, treated as in (b): the entire
envelope is heavily labelled with ferritin.
individual chemical components (Krause, 1972; Heyrner et al., 1973; Davis et al., 1973), then
the extraction of its various parts should result in the loss of any layer. However, the ultrastructural appearance of Group A and C streptococci walls after different chemical extractions showed that none of the procedures used resulted in the complete removal of any
morphologically distinct layer. These findings contrast with the observations of Swanson &
Gotschlich (1973) who found loss of the inner electron-dense layer after HN02 treatment
and concluded that this layer could be identified with the teichoic acid. Nor can this layer
represent the plasma membrane (or its part), as has been suggested by Huis in’t Veld &
Linssen (1973), (see Fig. If). Even with hot forrnarnide (Krause, 1963), which has been
proved to be the most effective method for extraction of polysaccharide and teichoic acids,
the triple-layered appearance of wall remnants remained unchanged. In agreement with
findings in Bacillus licheniformis (Millward & Reaveley, 1974), no simple quantitative
correlation between the loss of wall polymers and reduction in wall thickness could be
found, a finding which argues against the wall being constructed in concentric layers.
The use of irnmunoelectron microscopy allows a direct proof of the localization of
peptidoglycan. The interaction of .peptidoglycan of Group A and C streptococci with
peptidoglycan antibodies raised in rabbits followed by incubation with ferritin-labelled
anti-rabbit IgG resulted in heavy labelling on both sides of the peptidoglycan fragment.
Since the ferritin conjugate alone did not react, the reaction could be used for the specific
detection of peptidoglycan. The fact that on trypsin-treated walls peptidoglycan was
localized both on the inner and outer surface also argues against earlier models of the
streptococcal wall substructure in which peptidoglycan forms the innermost layer. Ferritin
particles were mainly bound on to filamentous structures which protruded from the surface
of both peptidoglycan and walls. These filaments were not observed in untreated samples.
Perhaps in the presence of specific antibodies these polymers are held in a native, extended
configuration, whereas in the course of fixation and dehydration of untreated cells they
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
M. W A G N E R , B. W A G N E R A N D M. RPC
292
become attached to the basal structure (Emyanitoff et al., 1976). In an attempt to explain
the nature of these filaments, experiments using antibodies against individual moieties of
the peptidoglycan molecule are in progress.
Peptidoglycan antibodies were also bound to the surface of whole streptococci. The
streptococci used for these experiments were free of Fc-binding factor (Christensen et al.,
1976), since the presence of Fc-binding factor disturbed the specificity of the antibody
binding. By phage-adsorption experiments Cleary et al. (1977) have shown that at least
part of the peptidoglycan is located at the coccal surface. Similar results has been obtained
by means of the immunofluorescence technique on B. licheniforrniscells (Hughes & Stokes,
1971).
The ferritiii particles on whole cocci were localized on filamentous protrusions similar to
those shown earlier to contain M protein (Swanson et al., 1969; Wagner & Wagner, 1972a)
and lipoteichoic acid (Beachey & Ofek, 1976). There is no evidence at present as to whether
these components are localized on the same filaments or different ones.
A specific interaction with fluorochrome-labelled lysozyme has been used to detect
peptidoglycan in bacteria (Gould et al., 1963). Recently, we localized peptidoglycan in the
walls of Group A and C streptococci electron-microscopically by means of peroxidaselabelled ly sozyme (Wagner & Wagner, 1978). Isolated peptidoglycan and trypsin-treated
walls showed strong labelling whereas the surface of cocci reacted only weakly. These
observations suggested that the lysozyme-sensitive bonds between N-acetylmuramic acid
and N-acetylglucosamine in the glycan are masked in whole organisms.
All the results presented above argue against the peptidoglycan forming an inner layer of
the streptococcal wall and suggest that it must be present throughout the wall as a network.
Evidently, the wall possesses a complicated mosaic structure in which the higher electron
density of some parts is due to a higher local concentration of some osmiophilic molecular
groups.
Since streptococcal walls have more chemical components than walls of most other Grampositive bacteria, a simple comparison of their substructure is not possible. Nevertheless,
studies in Streptococcus faecalis (Garland et al., 1975), Staphylococcus aureus and B. licheniformis (Millward & Reaveley, 1974) have also produced evidence for a mosaic arrangement
of the components in the wall of Gram-positive bacteria.
We are indebted to Dr J. HavliEek, Institute of Hygiene and Epidemiology, Prague, for
the estimation of the Fc-binding factor. The skilful technical assistance of Miss E. Bonnovi,
Mrs E. Chmhtalovk and Miss S. Kunze is gratefully acknowledged.
REFERENCES
ABDULLA,
E. M. & SCHWAB,
J. H. (1965). Immunofragment of IgG. Acta pathologica et micrological properties of bacterial cell wall mucobioiogica scundinavica BS4, 73-76.
peptides. Proceedings of the Society for Experi- CLEARY,
P. P., WANNAMAKER,
L. W., FISCHER,
M. &
mental Biology and Medicine 118, 359-362.
LAIBLE,N. (1977). Studies of the receptor for
ARCHIBALD,
A.R., COMES,H.E. & STAFFORD, phage A25 in group A streptococci: the role of
G. €3. (1969). The action of dilute alkali on
peptidoglycan in reversible adsorption. Journal of
bacterial cell walls. Biochemical Journal 113,
Experimental Medicine 145, 578-593.
899-900.
COLE,R. M. (1968). The structure of the Group A
BEACHEY,
E. H. & OFEK,I. (1976). Epithelial cell
streptococcal cell and its L-form. In Current
binding of Group A streptococci by lipoteichoic
Research on Group A Streptococcus, pp. 5-42.
Edited by R. Caravano. Amsterdam: Excerpta
acid on fimbriae denuded of M-protein. Journal
of Experimental Medicine 143, 759-771.
Medica Foundation.
CHEN,P. S., TORIBARA,
T. Y. & WARNER,
H. (1956). DAVIS,B. D., DULBECCO,
R., EISEN,H. N., GTNSMicrodetermination of phosphorus. Analytical
BURG, H.S., WOOD, W.B. & MCCARTY, M.
(1973). Microbiology, 2nd edn, pp. 708-726.
Chemistry 28, 1756-1758.
CHRISTENSEN,
P., JOHANNSON,
B. G. & KRONWALL, Hagerstown, Maryland: Harper and Row.
L. B. (1948). A specific
G. (1976). Interaction of streptococci with the Fc DISCHE,Z. & SHETTLES,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
Strep tococcal pep tidog ly can localization
293
color reaction of methylpentoses and a spectro- LANCEFIELD,
R. C. (1928). The antigenic complex of
photometric micromethod for their determination.
Streptococcus huemoIyticus. I. Demonstration of a
Journal of Biological Chemistry 175, 595-603.
type-specifk substance in extracts of Streptococcus
EMYANITOFF,
R. G., RUCINSKY,
T. E. & BIRDSELL, haernolyticus. Journal of Experimental Medicine
D. C. (1976). Electron microscopy of antibody47,91-103.
labelled cells of Streptococcus mutans. Cariadian LANCEFIELD,
R. C. (1943). Studies on the antigenic
composition of Group A hemolytic streptococci.
Journal of Microbiology 22, 891-895.
FAULK,
W. P. & TAYLOR,
G. M. (1971). An immunoI. Effect of proteolytic enzymes on streptococcal
colloid method for the electron microscope.
cells. Journal of Experimental Medicine 78,
465-476.
Immunochemistry 8, 1081-1083.
FULLER,A. T. (1938). The formamide method for LANCEFIELD,
R. C. & PERLMANN,
G. E. (1952).
the extraction of polysaccharide from haemolytic
Preparation and properties of type-specific M
antigen isolated from Group A, type 1 hemolytic
streptococci. British Journal of Experimental
Pathology 19, 130-1 39.
streptococcus. Journal of Experimental Medicine
96, 71-82.
GARLAND,
J. M., ARCHIBALD,
A. R. & BADDILEY,
J.
(1975). An electron microscopic study of the loca- LEVY,H. B. & SOBER,H. A. (1960). A simple
chromatographic method for preparation of
tion of teichoic acid and its contribution .to
gammaglobulin. Proceedings of the Society for
staining reactions in walls of Streptococcusjaecalis
Experimental Biology and Medicine 103, 250-252.
8191. Journal of General Microbiology 89, 73-86.
D. A. (1974).
GOULD,G. W., GEORGALA,
D. L. &HITCHINS,
A. D. MILLWARD,G. R. & REAVELEY,
(1963). Fluorochrome-labelled lysozyme: reElectron microscope observations on the cell walls
of some Gram-positive bacteria. Journal of Ultraagent for the detection of lysozyme substrate in
structure Research 46, 309-326.
cells. Nature, London 22, 385-386.
HEYMER,
B., SCHAFER,
H. & HAFERKAMP,
0. (1973). NAKANE,P. K. & PIERCE,G.B. (1966). Enzymelabelled antibodies : preparation and application
Vergleichende elektronenoptische und biochemfor the localization of antigens. Jownal of Histoische Untersuchungen iiber die Wirkung einer
chemistry and Cytochemistry 14, 929-93 1.
Streptomyces albus Endo-N-Acetylmuramidase
E. S. (1963). The use of lead citrate at
auf die Streptokokkenzellwand. Medical Micro- REYNOLDS,
high pH as an electron-opaque stain in electron
biology and Immunology 158, 193-208.
microscopy. Journal of Cell Biology 19, 208-212.
HILL,M. J. (1967). Action of bile salts on bacterial
RYTER,
A. & KELLENBERGER,
E. (1958). Etude au
cell walls. Nature, London 214, 1152-1 154.
microscope electronique de plasma contenan t de
HUGHES,
R.C. & STOKES,
E. (1971). Cell wall growth
l’acide deoxyribonucleic. I. Les nucleotides des
in Bacillus licheniformis followed by immunobacteries en croissance active. ZeitschriJt fur
fluorescence with mucopeptide-specific antiserum.
Naturforschung 13 b, 597-605.
Journal of Bacteriology 106, 694-696.
HUISIN’TVELD,J. H. J. & LINSSEN,
W. H. (1973). SALTON,M. R. J. (1964). The Bacterial Cell Wall,
p. 5 8 . Amsterdam: Elsevier.
The localization of streptococcal group and type
K. H. (1975). Chemical structure of the
antigens : an electron-microscopic study using SCHLEIFER,
peptidoglycan, its modifiability and relation to the
ferritin-labelledantisera. Journal of General Microbiologic activity. Zeitschrift fur Immunitatsbiology 74, 3 15-324.
forschung, experimentelle und klinische ImmunoKARAKAWA,
W. W., BRAUN,D. G., LACKLAND,
H.
logie 149, 104-117.
& KRAUSE,R. M. (1968). Immunochemical
K. H. & KANDLER,0. (1967). Zur
studies on the cross-reactivity between strepto- SCHLEIFER,
chemischen Zusammensetzung der Zellwand der
coccal and staphylococcal mucopeptide. Journal
S treptokokken. I. Die Aminosaiuresequenz des
of Experimental Medicine 128, 325-340.
Mureins von Str. thermophilus und Str. faecalis.
K A W W A , W. W. & KRAUSE,R. M. (1966).
Archiv fur Mikrobiologie 57, 335-364.
Studies on the immunochemistry of streptococcal
mucopeptide. Journal of Experimental Medicine SINGER,S. J. (1959). Preparation of an electrondense antibody conjugate. Nature, London 182,
124, 155-171.
1523-1524.
KRAUSE,R. M. (1963). Symposium on relationship
J. & GOTSCHLICH,
E. C. (1973). Electron
of structure of microorganisms to their immuno- SWANSON,
microscopic studies on streptococci. 11. Group A
logical properties. IV. Antigenic and biochemical
carbohydrate. Journal of Experimental Medicine
composition of hemolytic streptococcal cell walls.
138, 245-258.
Bacteriological Reviews 27, 369- 380.
J., Hsu, K. C. & GOTSCHLICH,
E. C.
KRAUSE,R. M. (1972). The streptococcal cell: re- SWANSON,
(1969). Electron microscopic studies on streptolationship of structure to function and pathococci. I. M antigen. Journal of Experimental
genesis. In Streptococci and Streptococcal Diseases.
Medicine 130, 1063-1091.
Recognition, Understanding, and Management,
pp. 3-18. Edited by L. W. Wannamaker and WAGNER,B. & WAGNER,M. (1972a). Immunelektronenmikroskopischer Nachweis von ZellJ. M. Matsen. New York and London: Academic
wandantigenen bei Streptokokken. 1. VerPress.
gleichende Darstellung des M-Proteins von
R. M. & MCCARTY,
M. (1962). Studies on
KRAUSE,
Streptococcus pyogenes mi t ferritin-, peroxydasethe chemical structure of the streptococcal cell
und ferrocenmarkierten Antikorpern. Zentralwall. TI. The composition of Group C cell walls
blatt fur Bakteriologie, Parasitenkunde, Infektionsand chemical basis for serologic specificity of the
krankheiten und Hygiene (Abteilung I ) 222,
carbohydrate moiety. Journal of Experimental
468-483.
Medicine 115, 49-62.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38
294
M. WAGNER, B. W A G N E R AND M. RqC
markierter Antikorper. Zentralblatt fur BakterioWAGNER,B. & WAGNER,M. (1975). Immunlogie, Parasitenkunde, Infektionskrarikheiten und
elektronenmikroskopischer Nachweis von ZellHygiene (Abteilung I ) 221, 100-105.
wandantigenen bei Streptokokken. 11. Der Nachweis des gruppenspezifischen Polysaccharids von WAGNER,M. & WAGNER,B. (1978). Die Verwendung von Lysozym-Peroxydase-Konjugaten
Streptokokken der Gruppe C mit ferritin- und
zum elektronenmikroskopischen Nachweis von
peroxydasemarkiertem Helix pomatia-Agglutinin.
Zentralblatt fur Bakteriologie, Parasitenkunde,
Peptidoglykan in der Zellwand von Streptokokken.
Zentralblatt fur Bakteriologie, Parasitenkunde, InInfektionskrankheiten und Hygiene (Abteilung I )
231, 81-91.
fektionskrankheiten und Hygiene (Abteilung I ) 240,
WAGNER,M., ROTH,J., WAGNER,B. & MEYER, 302-3 12.
K. E. (1957). Die KonH. W. (1974). Nachweis des M-Proteins von WOHLFARTH-BOTTERMANN,
trastierung tierischer Zellen und Gewebe im
Streptococcus pyogenes auf der Zelloberflache
Rahmen ihrer elektronenmikroskopischen Unterdurch Immun-Gefrieratzung. Zeitschrift fur
suchungen an ultradiinnen Schnitten. NaturAllgemeine Mikrobiologie 14, 61 1-616.
wissenschaften44, 287-288.
WAGNER,
M. & WAGNER,
B. (19723). Glutaraldehyd
als Kupplungsagens fur die Herstellung ferritin-
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 18:24:38