FEMS Microbiology Letters 18 (1983) 263-267
Published by Elsevier Science Publishers
263
Structural and chemical characterization of macromolecular arrays
in the cell wall of Bacillus brevis S 1
M i t s u k o A b e , M i t s u a k i K i m o t o a n d Z e n s a k u Yoshii
Departmentof Microbiology, Yamaguchi University School of Medicine, [/be, Yamaguchi-ken, Japan (755)
Received 28 January 1983
Accepted 31 January 1983
1. I N T R O D U C T I O N
Regularly arrayed macromolecular structures of
tetragonal or hexagonal symmetry in cell walls
have been found in m a n y bacteria [1-3]. In general, these structures are believed to be located in
the outermost layer of the cell wall, and consist of
monolayers of regularly arrayed macromolecules.
Each component of a regular array is composed of
a single homogeneous polypeptide, with carbohydrate as a minor constituent in some strains.
The multilayered surface structure in the outermost layer reported in a Gram-negative Spirillum
[8-11] is an exception. Exceptions for the location
of regular array are found in Gram-positive cells
of Clostridia [7] and Bacillus [6], which have monolayered regular arrays located not only in the
outermost layer but also in the innermost layer of
the cell wall. Among these exceptions, however,
few strains are known so far which have a multilayered composite array on both the outer and
inner side of the peptidoglycan network. In this
report we describe a strain of Bacillus brevis which
exhibits a unique multilayered composite array
(amorphous-hexagonal double layers) on both sides
of the peptidoglycan sheet.
2. M A T E R I A L S A N D M E T H O D S
2.1. Culture conditions. A strain of B. brevis
named S1, originally isolated in this laboratory
f r o m soil (details of the characterization:
manuscript in preparation), was grown in nutrient
broth at 30°C, aerobically.
2.2. Preparation of cell walls. Late logarithmic
cells were sonicated 4 times at 80 W for 1 min with
a Branson sonifier M185, and treated with DNase
(Boehringer Mannheim: 20 ffg/ml), R N a s e
(Sigma; 40 ~ g / m l ) and MgC1 z (5 mM) at 37°C
for 30 min. To remove cytoplasmic membrane, the
crude cell wall was treated with 1% Triton X-100
at room temperature for 20 min, and washed
several times with water by centrifugation.
2.3. Isolation and reassembly of outer and inner
layer. The washed cell wall preparation was treated
with 6 M urea or 2 M guanidine hydrochloride
(GuHC1) (1 h, room temperature, 10-20 mg protein/ml). The peptidoglycan sacculus was separated from the reaction mixture, by sedimentation at 34 000 x g for 30 min and, when necessary,
the precipitate (peptidoglycan sacculus) was
washed twice with water by the centrifugation.
After removal of the peptidoglycan, outer and
inner layers were reassembled from the supernatant by exhaustively dialysing against water.
Assembled materials were then pelleted by centrifugation at 20 000 x g for 20 min.
2.4. Lysozyme treatment of cell walls. The wall
preparation (1-2 m g / m l dry weight) was incubated with egg white lysozyme (Sigma; 500
ffg/ml), MgC12 (5 mM) in 50 m M phosphate, p H
7.4 for 3 h at 37°C. After incubation, the suspen-
0378-1097/83/0000-0000/$03.00 © 1983 Federation of European Microbiological Societies
264
Fig. 1. All bars represent 0.1 ~m. Negatively stained preparation of cell wall. Micrograph with a higher magnification is shown in the
right side. ATL, amorphous thin layer.
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sion was centrifuged at 12000 × g for 20 min at
4°C, and the precipitate washed twice with water.
2.5. SDS-PAGE. Solubilized preparations were
analyzed by SDS-PAGE in 5% gels [4], with R N A
polymerase (a, /3, /3' subunits), bovine serum albumin and trypsin inhibitor (Boehringer Mannheim) as M r standards. Duplicate samples were
electrophoresed, with one gel stained with
Coomassie brilliant blue and the other with PAS
[51.
2.6. Electron microscopy. Samples were negatively stained with 1% uranyl acetate and shadowed
with chromium. Thin-sectioned materials were
fixed by the double fixation m e t h o d of
glutaraldehyde-osmium tetraoxide. Electron micrographs were taken with a JEM-100B (JEOL)
operating at 80 kV.
cells (Fig. 3A) and cell walls (Fig. 3, B - D ) possessed two or three distinct electron-dense layers,
namely outer layer (OL), middle layer (ML) and
inner layer (IL). OL and ML were continuous and
the thickness of OL and IL were approx. 16 nm,
whereas IL was interrupted (Fig. 3B). OL and IL
contained periodic projections (with a center-tocenter spacing of approx. 17 nm) like spikes directed toward the ML (Fig. 3, A, C). The centerto-center distance of 17 nm observed in the hexagonally arrayed surface structure (Fig. 1) coincided
approximately with the center-to-center spacing of
the periodic projection shown in Fig. 3C. Thus,
these periodic projections seemed to correspond to
each of the subunit of hexagonally arrayed surface
structure. The pattern of hexagonal regular-arrays
were obvious in tangential sections (Fig. 3D).
3.2. Isolation and reassembly of outer- and
inner-layer proteins. By treatment of the cell wall
3. RESULTS
3.1. Morphological views of the cell wall. In
negatively stained preparations the entire surface
of the cell wall appeared to be covered by hexagonally arrayed structures with a center-to-center
spacing of 17 nm (Fig. 1). Each particle of the
regular array showed a doughnut shape with a
hole of 2-3 nm diameter (Fig. 1). The thin layer
approx. 2 nm thick; Fig. 1, ATL) observed around
the cell wall corresponds to the amorphous layer
in shadowed samples (Fig. 2). In shadowed specimens only an amorphous structure was visible,
and not the hexagonal array. This indicated that
the whole surface of the cell wall was covered by
an amorphous structure over the hexagonally regular structure. Thin sections revealed that whole
with 6 M urea or 2 M GuHC1, both OL and IL
disappeared entirely and only ML was observed in
thin-section samples (Fig. 4). In negatively stained
preparations, on the other hand, only a smooth
and thin sacculus was visible (Fig. 4). GuHC1- or
urea-solubilized fractions sheet structures consisting of two layers with a hexagonally arrayed and
an amorphous layer were reconstructed (Fig. 5).
The subunits of reassembled regular-array had the
same distance with each other as the original appearance in intact cell walls. These evidence indicates that both OL and IL are composed of two
macromolecular structures, which are a hexagonally arrayed layer and an amorphous layer.
3.3 Composition of outer and inner layer. Solubilized fractions obtained by GuHC1 and urea treatment of the cell wall gave the same electrophoretic
Fig. 2. Shadowed preparation of intact cell wall.
Fig. 3. Thin section of whole-cell and cell-wall preparation. (A) whole cell; (B) cell wall; (C) higher magnificationof cell wall sample.
OL, outer layer; ML, middle layer, IL, inner layer; (D) tangential section of cell wall.
Fig. 4. Negativelystained preparation and thin section of urea-extracted cell wall.
Fig. 5. Shadowed sample of reassembled preparation of urea extract.
Fig. 6. Shadowed preparation and thin section of lysozyme-treatedcell wall.
Fig. 7. SDS-PAGEof outer (OL) and inner (IL) layer of cell wall. 1, Mr standards; II, urea extract; III, GuHC1 extract.
266
patterns by SDS-PAGE (Fig. 7). They gave two
polypeptide bands with M r values of 129000 and
107000. The M r 129000-band was always wider
than the M r 107000 band. Both bands stained
with PAS (not shown) consistent with the presence
of carbohydrate.
3.4. Lysozyme treatment of cell walls. When the
cell walls were hydrolyzed completely with lysozyme, cell wall sacculus structures were easily disrupted (data not shown) and outer- and inner-layer
fragments remained. When cell walls were treated
with lysozyme using milder conditions, they were
destroyed partially and in section exhibited incomplete disappearance of the M L (Fig. 6). These
observations indicate that ML is a lysozyme-susceptible peptidoglycan layer.
4. D I S C U S S I O N
Three morphological features of cell wall were
revealed in B. brevis S1. (a) The cell wall had three
(in some areas two) electron-dense layers. This
finding is important because typical cell walls of
Gram-positive bacteria have a single, fairly homogeneous, electron-dense layer, 15-80 nm [2] or
20-50 nm [3] thick. (b) The macromolecular structure of each of the two electron-dense layers described above were multiple composite layers constructed from a hexagonally arrayed layer and an
amorphous layer adhering closely to each other.
(c) The multiple composite arrays were located in
both the OL and IL part of the three electron-dense
layers (OL, ML, IL) in the cell wall of this strain.
In most cases, regularly arrayed macromolecular
structures are believed to be located only on the
outer surface of the cell wall [1-3]. However, in a
few Gram-positive bacteria, such as B. polymyxa
[6], Clostridium thermohydrosulfuricum [7] and Cl.
thermosaccharolyticum [7], regularly arrayed layers
were observed both as the outermost surface of the
cell and also in the innermost cell-wall layer, just
outside the cytoplasmic membrane. In Clostridia
[7], this phenomenon was inferred to occur after
the cytoplasmic membrane became detached from
the peptidoglycan and the structural folding of
subunits of the regular array took place immediately outside the cytoplasmic membrane and
the subunits assembled into regular arrays inside
the peptidoglycan layer. An inner layer of the cell
wall of B. brevis SI, was observed frequently, even
in the place where plasmolysis did not occur (Fig.
3A). Thus the proposed mechanism for the construction of IL in Clostridia species cannot be
applied to the IL of B. brevis S I. Further studies
seem to be necessary for a better understanding of
IL in this strain.
The presence of multiple regularly arrayed layers
were observed in some Spirilla sp. [8-11] and
Nitrosocystis oceanus [12]. These multiple regular
arrays of Gram-negative strains, located only in
the outermost layers, appeared to be very complex,
possessing two or more stacked layers with hexagonal, tetragonal, linear, and amorphous areas.
A m o n g Gram-positive bacteria, a similar structural arrangement has been seen only in B. brevis
47 [14] and S1. The OL and IL appear to contain
two glycoproteins of M r 129000 and 107000
(Fig. 7). The thickness of the amorphous layer and
OL were 2 nm and 16 nm, respectively (Fig. 1).
This means that the volume of the regularly arrayed
layer is larger than the amorphous layer. Although
further quantitative analyses are necessary to define the volume of the hexagonally arrayed and
amorphous structures, because of the relative
amounts present (Fig. 1) we believe that the M r
129 000 protein constitutes the hexagonally arrayed
layer and the Mr 107000 protein the amorphous
layer.
The major part of our work has been reported
in 1981 [13]. Another group recently [14] reported
that B. brevis 47 had a multiple-composite layer
and the structure of its cell wall resembled that of
our strain, B. brevis S1. Characteristics of B. brevis
S1 and B. brevis 47 are compared in Table 1. The
sizes and M r values of cell wall subunits are different. The B. brevis S1 subunit appears to contain
carbohydrate, and the multiple-regular arrays
(especially OL) were a constitutive structure in our
strain. B. brevis 47, on the other hand, shed both
IL and OL into the medium, in large amounts.
during growth [14]. Although differences in characteristics of regular-array components were obvious between the two strains, the multiple-composite array in the cell wall may be a ubiquitous
structure of B. brevis. In the contex of this hy-
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Table 1
Comparison of cell wall components between B. brevis SI and B. brevis 47 [14]
Strain characteristics
B. brevis S1
B. brevis 47
Constituent 1
Constituent 2
hexagonal (C-C 17 nm)
amorphous
hexagonal (C-C 14.5 nm)
amorphous
Composition
Hexagonal
Amorphous
glycoprotein (M r 129000)
glycoprotein ( M r 107 000)
protein (M r 150000)
protein (M r 130000)
Localization
both sides of PG
both sides of PG
Stability
stable
( OL is completely ]
constitutive
J
unstable
(shed into medium)
C-C, center-to-center spacing; PG, peptidoglycan layer.
pothesis, the observed difference between the above
two strains in the stability of IL and OL may
reflect (1) the higher activity of autolysing enzymes of strain 47 for the cleavage and excreation
of IL and OL, or (2) the contribution of sugar
component of strain S1 for the stability of IL and
OL.
ACKNOWLEDGEMENT
We are indebted to Dr. Takahiro Nishimune for
helpful discussions and suggestions.
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