The primary goal of this review is to provide a compilation of the complex architectural features of staphylococcal cell walls and of some of their unusual morphogenetic traits including the utilization of murosomes and two different mechanisms of cell separation. Knowledge of these electron microscopic findings may serve as a prerequisite for a better understanding of the sophisticated events which lead to penicillin-induced death. For more than 50 years there have been controversial disputes about the mechanisms by which penicillin kills bacteria. Many hypotheses have tried to explain this fatal event biochemically and mainly via bacteriolysis. However, indications that penicillin-induced death of staphylococci results from overall biochemical defects or from a fatal attack of bacterial cell walls by bacteriolytic murein hydrolases were not been found. Rather, penicillin, claimed to trigger the activity of murein hydrolases, impaired autolytic wall enzymes of staphylococci. Electron microscopic investigations have meanwhile shown that penicillin-mediated induction of seemingly minute cross wall mistakes is the very reason for this killing. Such "morphogenetic death" taking place at predictable cross wall sites and at a predictable time is based on the initiation of normal cell separations in those staphylococci in which the completion of cross walls had been prevented by local penicillin-mediated impairment of the distribution of newly synthesized peptidoglycan; this death occurs because the high internal pressure of the protoplast abruptly kills such cells via ejection of some cytoplasm during attempted cell separation. An analogous fatal onset of cell partition is considered to take place without involvement of a detectable quantity of autolytic wall enzymes ("mechanical cell separation"). The most prominent feature of penicillin, the disintegration of bacterial cells via bacteriolysis, is shown to represent only a postmortem process resulting from shrinkage of dead cells and perturbation of the cytoplasmic membrane. Several schematic drawings have been included in this review to facilitate an understanding of the complex morphogenetic events.
INTRODUCTION
Microbiologists are highly
interested in the sophisticated, unique architecture and morphogenesis
of the cell wall of staphylococci which make these bacteria suitable
for exploring the reason for penicillin-induced death during defined
morphogenetic steps (48, 50, 53). More detailed knowledge of
those structural "weak points" in the staphylococcal wall, which
turned out to be the main sites of penicillin action, is an important
prerequisite not only for attempts to enhance the efficiency of
beta-lactam antibiotics but also for efforts to attack even
staphylococci that are highly resistant to this type of antibiotic (the
so-called methicillin-resistant Staphylococcus aureus
[MRSA] strains). Great attention is being paid to structural and
chemical variations in the cell walls of such highly resistant strains
(23, 24, 74, 87) and to factors involved in the biosynthesis
of those staphylococcal cell walls, both of which might be suitable as novel targets in the combat against MRSA (70). Such
extremely drug-resistant strains of S. aureus are already
posing major public health problems (21). Many physicians
are gravely concerned about such antibiotic resistance, and they are
highly interested in any attempts to overcome this problem
(6).
Therefore, this review shall serve several aims. First, we want to
compile our current knowledge of the macromolecular wall architecture
and wall morphogenesis of staphylococci. On the basis of our last
review (42) and new data we will discuss some recent concepts including even some speculative considerations on
staphylococcal cell wall morphogenesis, wall degradation, and the
combination of both these processes during cell separation. In
particular, we have paid great attention to all the morphologic and
morphogenetic details of the staphylococcal cell walls which, some day,
might serve as new targets for the badly needed progress in our
therapeutic efforts. However, since recent reviews are available
concerning biochemical data (see references 78 and
118), only the most relevant findings will be
mentioned and all details are purposely omitted.
Second, our contribution will deal with penicillin-induced structural
variations during staphylococcal cell wall morphogenesis and
degradation. Since it has been shown that staphylococci do not die from
bacteriolysis but from very characteristic cross wall defects
(50-53), we will focus our interest (i) on those
morphogenetic wall variations which regularly lead to death and (ii) on
attempts of the staphylococci to survive in spite of such morphological handicaps.
In order to update the review, some electron micrographs from rather
early publications have been replaced by more recent high-resolution
pictures and several other, unpublished ones have been included (from
our archive, which now holds about 60,000 electron micrographs of
staphylococci). In order to prevent fixation artifacts, electron
microscopic pictures of unfixed, freeze-fractured staphylococci are
included as often as possible. They are regarded as "images of latent
living bacteria."
We hope that the ample schematic drawings will help give an idea of the
highly differentiated dynamic processes of wall morphogenesis, wall
degradation, and fatal wall variations. These simplified, line art
illustrations are not only a visual aid for the reader but they also
give reliable information to those scientists who do not have time
enough to read all the details of this review. That is why we made
every effort to include in the schematic drawings all of the data which
seem to be essential for an overview.
Furthermore, recent unpublished findings on staphylococcal wall
morphology and morphogenesis have been included here with a view to
presenting state of the art knowledge about a fascinating field: the
staphylococcal cell wall.
Since detailed knowledge of wall morphogenesis is the most important
prerequisite for analyzing the very complex sequences of the
penicillin-induced killing process, this review is divided in two
parts: "Morphogenesis of the staphylococcal cell wall" and
"Penicillin-induced death."
MORPHOGENESIS OF THE STAPHYLOCOCCAL CELL WALL
Chemical Composition of the Staphylococcal Cell Wall
Chemical structure of the cell wall of S. aureus.
The cell wall of S. aureus shows the typical features
of gram-positive bacterial cell walls. Under the electron microscope it
appears as a relatively thick (about 20 to 40 nm) homogeneous structure.
The chemical structure of its major component, the peptidoglycan, has
been known for a long time (see reference 115).
This heteropolymer consists of a disaccharide backbone formed by
alternating 
-1-4-N-acetylglucosamines and
N-acetylmuramic acids. The average chain length is in the
range of 10 disaccharides (119). Tetrapeptides consisting of
L-alanine, D-glutamine, L-lysine,
and D-alanine are attached to the
N-acetylmuramic acid. About 90% of these stem peptides are
cross-linked to the stem peptides of another glycan chain by a
pentaglycine group (74). This pentaglycine is a
characteristic feature of the staphylococcal peptidoglycan and connects
the 
-amino group of the L-lysine of one stem peptide to
the D-alanine of the other one. The stem peptides which are
not cross-linked carry an additional D-alanine which is
cleaved during the cross-linking reaction. The structure of the
staphylococcal peptidoglycan is summarized in a schematic drawing (Fig.
1).
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FIG. 1.
The structure of peptidoglycan and the sites where
peptidoglycan may be attacked by cell wall hydrolases. Three glycan
strands of peptidoglycan, consisting of alternating
N-acetylmuramic acid and N-acetylglucosamine are
depicted. The tetrapeptides (stem peptides), branching from
N-acetylmuramic acid, are interconnected by pentaglycine
bridges. The sites where cell wall hydrolases may attack peptidoglycan
are indicated by arrows, but staphylococci contain only three of these
wall hydrolases (amidase, glucosaminidase, and endopeptidase).
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The process of cell wall cross-linking is catalyzed by transpeptidases,
the penicillin-binding proteins (PBPs) (74). Knowledge about
the exact functions of the four staphylococcal PBPs is not as detailed
as is what is known about the PBPs in Escherichia coli, but
there is evidence that the function of PBP 1 is the most important one
for the survival of staphylococci exposed to beta-lactams (8,
112). PBP 4, in contrast, seems to be responsible for secondary
cross-linking, as can be deduced from a low cross-linking rate in PBP
4-defective S. aureus mutants (58).
The so-called PBP 2a, the reason for methicillin resistance in
staphylococci, seems to need proper pentaglycine interpeptide bridges
to perform cross-linking reactions (70). Mutant strains with
shortened interpeptide bridges (femA-femB, containing
significantly increased amounts of mono-, di-, and triglycine residues)
showed a drastically reduced resistance level (24, 25, 58, 70, 87) and a reduced level of cross-linking when grown in the
presence of beta-lactam antibiotics (23, 74).
O acetylation of the muramic acid is another important feature of the
staphylococcal peptidoglycan (114). Due to this,
staphylococcal cell walls are rarely degraded by lysozyme, which is
sterically hindered in its action (62).
About 50% of the total mass of the cell wall consist of teichoic acid,
a polymer covalently linked to the muramic acid via phosphodiester
bonds. Teichoic acids consist of long chains of ribitol phosphate units
(114); they are usually replaced by ester-linked D-alanine (28). The degree of such substitution
seems to have a very great effect on the activity of autolytic enzymes
(29).
Cell wall hydrolases of S. aureus.
The necessity
that bacteria with a compact peptidoglycan network have their own cell
wall hydrolases is quite evident. In order to divide and separate, the
cells must cleave certain parts of their walls in a highly regulated
manner (for a review, see reference 118).
Disturbance of these control mechanisms usually leads to cell lysis;
this is the reason why endogenous cell wall hydrolases are called wall
autolysins or autolytic wall enzymes. Cell wall hydrolases are also a
prerequisite for cell wall morphogenesis and turnover, a problem to be
discussed in more detail in a following section.
S. aureus has three different autolytic enzymes: an
N-acetylglucosaminidase, an N-acetylmuramidase,
and an endopeptidase (114, 123). The sites where these
enzymes attack the staphylococcal peptidoglycan are shown in Fig. 1.
However, examination of cell wall hydrolases by the so-called zymogram
method has shown (via Triton-mediated reactivation of autolysins) that
several bands are capable of hydrolyzing peptidoglycan (61, 63,
90), indicating that these autolytic activities must be
represented by more than three enzymes. The number of bacteriolytic
enzymes, however, decreases when staphylococcal cells reach the
stationary phase (69). The overall rate of the murein
hydrolase activity seems mainly to be regulated genetically (by the
lytS-lytR regulatory locus) (16).
Recently, the atl gene encoding an autolytic enzyme with
bifunctional activities was cloned and sequenced (102). The
two domains contain an
N-acetylmuramyl-L-alanine-amidase (AM) and an
N-acetylglucosaminidase (GL). A gene for an additional
amidase, encoding a polypeptide with a molecular weight of 23,000, was cloned earlier (61).
The two cell wall lytic enzymes AM and GL proved to be capable of
acting as cluster-dispersing enzymes (see "Inhibition of cell
separation results in the formation of pseudomulticellular staphylococci") when externally added to cluster-forming mutant strains of S. aureus (20, 122).
Purification and production of antibodies against these autolysins
enabled immunoelectron microscopic investigations revealing the exact
localization of these enzymes. They were shown to be arranged in a
circumferential double ring at the surface of the peripheral cell wall
(146); after penicillin treatment these enzymes could be
detected at the strictly localized perforations of the peripheral wall
(123) which initiate cell separation (see "Initiation of
cell separation via murosomes").
The physiological role associated with the staphylococcal endopeptidase
activity is still unclear. It has been speculated that endopeptidase
activity is needed for the completion of cell separation
(58).
Lysostaphin, an endopeptidase produced by Staphylococcus
simulans subsp. staphylolyticus is known to be an
effective agent for the complete lysis of S. aureus cell
walls. Whether the endopeptidases, possibly for example the gene
product of lytM (110), of S. aureus are capable of performing similar actions or whether they are only
needed for localized alterations of the peptidoglycan remains to be resolved.
Cell Division in Staphylococci
Differentiation of three consecutive division planes.
Different types of cross wall formation have been reported for
bacterial cocci. (i) In the division of Streptococcus
cells only simple pairs or chains are formed, like in rod-like
bacteria, indicating the existence of one single division plane. (ii)
In other cocci, for instance, in Pediococcus
(34), Thiopedia (104), Lampropedia (97), and possibly also
Deinococcus (99), successive division always
leads to four cells being arranged in two-dimensional tetrads. Later on
even, square tablets of 16 to 64 cells are formed, indicating the
existence of two division planes which during subsequent cell divisions
must regularly alternate their direction at right angles to each other.
(iii) In Sarcina (18) and the cyanobacterium Synechocystis (113), eight cells are arranged in
three-dimensional, cuboidal packets via stringent alteration of three
consecutive division planes.
For a long time it had not been evident to which of these three types
of cell division the staphylococci would belong, because complete cell
separation normally takes place right after cell division, resulting in
groups of individual cells; the very name staphylococci ("bunch of
cocci") already points to this characteristic formation of cell
groups. Only by scanning electron microscopy did it became evident that
the staphylococci do not belong to organisms with only two division
planes, as had been assumed earlier (42), but must be
ascribed to the Sarcina type. Their three-dimensional arrangements to eight-cell packets was demonstrated in some strains of
S. aureus (71); in most strains, however, this
was only possible after experimentally retarded cell separation (Fig.
2a).
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FIG. 2.
Scanning electron micrograph (a) and thin sections of
staphylococcal cells (b to f). (a) A packet of eight staphylococcal
cells was induced by liquoid; this packet is derived from one bacterium
by three consecutive cell divisions, each having changed its direction
at an angle of 90° to the preceding division plane. The three
division planes are indicated by arrows (reproduced with permission
from reference 139). (b) Characteristic alternation
of consecutive division planes (arrowheads) (reproduced with permission
from reference 41). (c) Asymmetrical initiation of
cross wall formation (arrowhead), Sp, splitting system (reproduced with
permission from reference 41). (d) Centripetal
growth of the closing cross wall. The splitting system (Sp) appears as
a darker central cross wall layer. (e) At the cell periphery, above the
closed cross wall with its splitting system (Sp), there is one of the
murosomes (MuS) (reproduced with permission from reference
50). (f) After a 2-h exposure to the antibiotic
batumin (1 µg/ml) the peripheral wall appears to be differentiated
into an outer layer, the so-called primary wall (prW), and an inner
layer, the so-called secondary wall (scW). The dark line between these
two layers represents the so-called stripping system (Str) of the
staphylococcal cell wall which is involved in cell wall turnover. The
remnants of the cutting through of the primary wall, the so-called
clefts, are marked by arrows.
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The characteristic alteration of consecutive division planes was
demonstrated in thin sections (Fig. 2b). However, the reason for this
astonishing alternation is far from being understood.
Neoformation of the cross wall.
Cross wall formation is
initiated asymmetrically (Fig. 2c) at one single starting point beneath
the peripheral cell wall (41). Like in other prokaryotic
cells, cross wall formation proceeds via centripetal growth resembling
a closing iris (Fig. 2d), until the tips of the ingrowing cross wall
eventually fuse in the center of the cell (Fig. 2e).
After closure of the cross wall, the peripheral cell wall in most cases
appears as a rather compact, homogeneous-looking structure (cf. Fig. 2e
and 3A). Treatment with various
antibiotics revealed, however, that it consists of two layers (Fig.
2f): (i) an outer layer, the so-called primary wall, and (ii) an inner
layer, the so-called secondary wall (42). The secondary wall
continues into the cross wall (Fig. 2f and 3B). Sandwiched between the
primary and the secondary walls is the so-called stripping system that is involved in cell wall turnover (see "Wall thickening via
underlayering processes"). Sometimes, characteristic "clefts" are
left behind on the cell surface after the cutting through of the
primary wall during early stages of cell separation (cf. Fig. 2f, and
3C and see "Cell separation in staphylococci"). If they are not
turned over (Fig. 13b), clefts are capable of marking, even during
later stages of the cell cycle, the site where the primary wall had first been cut through (135).
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FIG. 3.
Nomenclature of the different parts of the
staphylococcal cell wall. Schematic overview of the common parts of the
cell wall, as seen in the electron microscope by investigating thin
sections of fixed staphylococci. (For more details see Fig. 18). (A)
Cell wall, splitting system, and murosomes. A highly elastic peripheral
cell wall (pW) protects the protoplast against the extremely high
turgor of the cytoplasm. The cross wall (cW) contains the splitting
system (Sp) consisting of concentrically arranged ring-shaped tubuli.
The splitting system is involved in cell separation. Minute, vesicular,
extraplasmatic wall organelles, the murosomes (MuS), are located in two
circumferential rows above the closed cross wall. They are engaged in
lytic processes during cross wall formation and initiation of cell
separation. Reference figure, Fig. 2e. (B) Primary and secondary walls.
The seemingly homogeneous peripheral cell wall is, in fact,
differentiated into an outer layer (the so-called primary wall [prW])
and an inner layer (the so-called secondary wall [scW]). The
secondary wall, which continues into the cross wall, is deposited
beneath the primary wall in connection with the formation of a new
cross wall. The dark line between the primary and the secondary wall
represents the so-called stripping system (Str), which is involved in
wall turnover. Reference figures, Fig. 2f and Fig. 4a. (C) Clefts.
During early stages of cell separation the lytic capacity of the
murosomes is activated. The murosomes perforate and, subsequently, cut
through the primary wall, sometimes leaving behind characteristic
clefts (Cl) on the cell surface. If such clefts are not turned over,
they mark the site of cutting through even during later stages in the
cell cycle. Reference figures, Fig. 2f and 13b (D) Longitudinal slit of
the cross wall. Sandwiched between layers of the cross wall, a
preformed, longitudinal slit (Sl) is found in the center of the cross
wall, which contains the concentrically arranged tubuli of the
splitting system. If the splitting system is removed, the cross wall
layers are moved aside, exposing the container-like slit of the cross
wall. Reference figure, Fig. 4g.
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In the presence of penicillin (0.1 µg/ml), characteristic variations
were sometimes found in the "staining" of the wall material that
had been formed. The various parts of the wall structures reacted
differently to the uranium and lead salts applied to raise the contrast
for electron microscopy. The central region of the cross wall known to
be lysed during cell separation (54) (see "The lytic type
of cell separation") and the primary, peripheral wall both showed a
strikingly low electron density, while the newly underlayered secondary
wall and the future cross wall of the daughter cells produced under the
influence of penicillin were intensely stained (Fig.
4a). Such different staining effects of
the two parts of the cross wall not only indicated the existence of
different wall qualities in this region of the cell wall but also
proved to be helpful for analyzing the sequence of some morphogenetic events (see "Murosomes and their role in cross wall
morphogenesis").
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FIG. 4.
Thin sections of staphylococcal cells. (a) After
treatment with penicillin (0.1 µg/ml) the secondary wall (scW) is
intensely stained while the primary wall and the central parts of the
cross wall (the so-called transitory cross wall material) are hardly
stained. A murosome (MuS) is detectable in the secondary wall. (b) This
section, running exactly through the middle of the cross wall, reveals
the concentrically arranged tubuli of the splitting system. The
hexagonally shaped inner edge of the closing cross wall is marked with
arrows (reproduced with permission from reference
41). (c) The diameter of the tubuli of the splitting
system enlarges continuously during growth in the presence of spermine
(arrowheads). (d) Treatment with Triton X-100 likewise resulted in an
enlargement of the tubular structures of the staphylococcal splitting
system (Sp). (e) By extraction of the LTA, the splitting system
disappeared. (f) Isolated cell wall of a staphylococcus after removal
of the cytoplasm. The splitting system is still detectable (arrows)
(reproduced with permission from reference 78). (g)
Extraction of LTA from the isolated cell wall leads not only to the
disappearance of the splitting system but also to a premature
separation of the central cross wall region (arrows) (reproduced with
permission from reference 78).
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The most distinctive feature in the center of the nascent cross wall
was a thin electron-dense layer (Fig. 2d and e); since this layer
proved to be involved in cell separation, we have called it the
splitting system of the cross wall (41). This splitting system (Fig. 4b) is 7 to 10 nm wide (103) and has been shown to consist of a concentrically arranged system of about 14 to 18 ring-shaped tubuli, each 7 to 10 nm in diameter (41). A
similar concentric ring system has been found to be located in the
cross wall of the cyanobacterium Phormidium uncinatum
(33). Early data concerning the chemical nature of this
cyanobacterial system indicated that it does not consist of
peptidoglycan (33).
In staphylococci, the splitting system can be influenced by growth
conditions; in the presence of spermine, the diameter of the tubuli was
continuously inflated (up to 20 nm) (Fig. 4c). Treatment with Triton
X-100 enlarged the width of the splitting system slightly, but it
caused a considerable increase of its electron density (Fig. 4d), thus
indicating its composition of tubular structures. At the same time, the
layer directly beneath the cell wall, the so-called membrane-wall
interlayer (see Fig. 6h and i), was affected.
No convincing data are so far available about the chemical composition
of the staphylococcal splitting system; teichoic acid-like material,
the lipoteichoic acid (LTA), has been assumed to be associated with
this system (96, 125, 132). In fact, LTA extraction of
staphylococci via the phenol method (28) led to the
disappearance of the splitting system (Fig. 4e). It is highly
interesting to note that extraction of teichoic acid from isolated cell
walls of S. aureus resulted not only in the disappearance of
the splitting system (cf. Fig. 4f and g) but also in premature
separation of the cross walls of the presumed daughter cells
exclusively within the region of the concentrically arranged rings of
the splitting system (78).
This setting apart of cross wall layers after extraction of teichoic
acid was always restricted to the region of the splitting system
without affecting the peripheral cross wall. These data suggest that
the splitting system can no longer be regarded simply as a special
chemical entity which is located in the compact cross wall; it should
be seen as a distinct layer fitted into a preformed longitudinal
container-like slit of the cross wall (Fig. 3D). We have to consider,
therefore, that the completed cross wall consists of different parts,
including a central slit-like container in which the concentrically
arranged tubuli of the splitting system are located and the rather
homogeneous-looking layers of the neighboring cross wall. These
findings are important for all considerations concerning the
involvement of the splitting system during mechanical cell separation
(see "An alternative, mechanical type of cell separation using the
splitting system of the cross wall").
Concerning the origin of the splitting system, no convincing data are
presently available. The possibility that we are dealing with a direct
extension of the cytoplasmic membrane can, however, be excluded since
the characteristic appearance of this membrane (see Fig. 6g and h) has
never been demonstrated. Several indications led us to presume that the
so-called membrane-wall interlayer (43), located between the
cytoplasmic membrane and the cell wall proper (see Fig. 18, MWI), is
involved in the formation of the splitting system. This membrane-wall
interlayer, in which minute hexagonally arranged particles are embedded
(see Fig. 6h and i), regularly covers the staphylococcal cytoplasmic
membrane (43). As discussed below the membrane-wall
interlayer is, probably, also involved in the formation of the
murosomes (see "Murosomes and their role in cross wall
morphogenesis").
Murosomes and their role in cross wall morphogenesis.
Minute vesicular structures, 30 to 40 nm in diameter, can be
observed in the peripheral cell wall above closed cross walls (Fig.
2e). Often, these structures appear in pairs located in the peripheral
wall directly above a closed cross wall (54). We named such
transparent, extraplasmatic wall organelles "murosomes" (38,
48, 50); they are capable of performing divers lytic activities
in the cell wall material. Murosomes above closed cross walls were
demonstrated most clearly in very thin sections of slowly growing and
dividing control cells, especially during the so-called stationary
phase of growth in which staphylococci exhibit relatively thick cell walls.
Pairs of such minute wall organelles were also traceable in the
peripheral cell wall at sites where a new cross wall was initiated (Fig. 5a). Freeze-etching in the presence
of sucrose or sodium chloride revealed that the murosomes are enclosed
by a definite envelope (Fig. 5b).
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FIG. 5.
Staphylococcal cells after thin sectioning (a and c to
e) or freeze fracturing in the presence of sucrose (b and f) or sodium
chloride (g). (a) A pair of murosomes (MuS) is located at the site of a
new cross wall initiation. (b) The murosomes (MuS [arrows]) appear to
be enclosed by a definite envelope (reproduced with permission from
reference 50). (c) Flattened or collapsed vesicular
murosomes (arrows) in the peripheral cell wall. (d) For initiation of
cross wall formation, the murosomes in both the just-separating
daughter cells have been anchored in the secondary wall where they
induced centripetal lytic processes which cut the secondary wall. Me,
membranous body. (e) Higher magnification of panel d showing details of
the new cross wall initiation. At the free ends of the secondary wall
created by the lytic activity of the murosomes the first assembling of
wall material for the formation of the new cross wall can be seen. A
local invagination of the cytoplasmic membrane, the membranous body
(Me), consisting of an envelope and a core, is associated with the site
of cross wall initiation. (f) A vesicular murosome exhibiting a tubular
tail-like extension (arrow) is detectable; this extension connects the
extraplasmatic murosome with the surface of the cytoplasm (reproduced
with permission from reference 49). (g) The
fracturing has exposed an envelope and a vesicular part of the murosome
and its tail. At the end of the tail-like extension of the murosome, a
ring-like structure is revealed which marks the connection site between
the murosome and the cytoplasmic membrane (CM). W, wall.
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In other control cells, however, instead of vesicular murosomes
rather flat structures only 10 to 15 nm wide were found, resembling more or less collapsed vesicular murosomes (Fig. 5c).
Since the initiation of a new cross wall is a very rapid process in
staphylococci, taking place within a few minutes from the logarithmic
phase of growth, only slowly growing cells from the stationary-growth
phase were suitable for analyzing cross wall morphogenesis.
Furthermore, advantage was taken of the fact that staphylococci
regularly alter their division plane and can therefore start cross wall
formation in the second division plane while cell separation is still
going on along the first division plane (Fig. 2b). In this way,
initiation of cross wall formation could be followed at the same time
in both the just-separating daughter cells (Fig. 5d and e). The
murosomes of both daughter cells were always found to be symmetrically
arranged to each other, indicating the possible existence of a
synchronized starting process for the cross wall initiation in both
daughter cells.
For initiation of a new cross wall, the murosomes always induced a
centripetally directed cutting of some inner layers of the peripheral
wall, probably the entire secondary wall (Fig. 5d and e and cf. Fig.
19d and f). It is speculated that after the cutting through of the
secondary wall its "free ends" function as assembling sites of
newly synthesized wall material for the formation of the new cross wall
(see Fig. 7).
High-resolution freeze-etching in the presence of sucrose or sodium
chloride (49) revealed the existence of vesicular murosomes with a tubular "tail-like" extension (Fig. 5f and g) which,
probably, are involved in this cutting process. The characteristic
result of these cutting processes was a direct connection between the extraplasmatic murosomes and the cytoplasmic surface; a local invagination of the cytoplasmic membrane was often observed beneath these connection sites (Fig. 5e). Sometimes a more-or-less ring-like structure was detected at the connection site of the murosomal tail
with the cytoplasmic surface (Fig. 5g). It is, however, still an open
question whether the tail of the murosome is in fact a structural
peculiarity of these organelles, engaged in cross-wall initiation, or
whether it is nothing but a sort of canal within the compact wall
material created by the lytic activity of the murosome. Furthermore, no
explanation has been found so far as to why at this stage of the cell
cycle the lytic activities of the murosomes are always directed
centripetally; one can only speculate that at this stage the outer
layers of the peripheral wall are more resistant to lytic processes of
this type than the inner layers.
Linearly arranged vesicular murosomes, during the onset of cross wall
formation on the inner surface of the peripheral cell wall (Fig.
6a), were also demonstrated by such
freeze-etching; probably, they were located in the region between the
cell wall and the cytoplasmic membrane. Interestingly, the
centripetally directed side of some of these murosomes proved to be
open (Fig. 6b). It is, however, still unknown whether these openings in
the "bottom" of the murosomes can be ascribed to the tail-like
extensions of those murosomes involved in the cutting of lower layers
of the peripheral wall (Fig. 5f and g).
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FIG. 6.
Freeze fractures of staphylococci in the presence of
sodium chloride (a and b) or normal freeze fracture (i) and thin
sections (c to h) of staphylococcal cells. (a) A row of linearly
arranged vesicular murosomes (MuS) on the concave fracture plane (EF).
(b) Murosomes (MuS) on the concave (EF) and convex (PF) fracture planes
of the cytoplasmic membrane are in different stages of maturation
(arrowheads); a ring-like structure is visible in the upper murosomes.
(c) In the presence of penicillin (0.1 µg/ml) the underlayered wall
material of the secondary wall reveals a higher contrast than the
primary wall. Two murosomes (arrows) embedded in the highly contrasted
layer are located beneath the primary wall. (d) In the presence of
penicillin (0.1 µg/ml) the two murosomes differentiate the nascent
cross wall into three parts, a central sector (white arrow) and two
lateral ones (black arrows)  , transparent "lytic" region at the
tip of the nascent cross wall. (e) In the presence of penicillin (0.1 µg/ml) the nascent cross wall is divided in three parts by the lytic
activity of murosomes (arrows). (f) Even in the presence of
trimethoprim (3.13 µg/ml) the murosomes for the second division plane
(arrows) are located at a 90° angle to the first division plane. (g)
Higher magnification of panel f. The murosomes (arrows) are located
outside the protoplast, between the cell wall and an invagination of
the cytoplasmic membrane. (h) In the presence of trimethoprim (3.13 µg/ml) the murosomes located between the primary wall and the
cytoplasmic membrane (CM) are covered with particles (white arrows)
probably derived from the membrane-wall interlayer (MWI) of the
cytoplasmic membrane. (i) Hexagonally arranged particles of an
isolated cytoplasmic membrane with its membrane-wall interlayer of a
control cell (reproduced with permission from reference
43).
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However, neither thin sections nor freeze-etchings of control cells
could answer questions concerning the exact localization of the
murosomes within the primary or secondary peripheral walls before the
cutting processes for cross wall initiation started. This was only
possible after the application of penicillin, by means of which we
became able to differentiate between primary and secondary wall
material (Fig. 4a). After treatment with this antibiotic the murosomes
appeared to be somewhat inflated (Fig. 6c), but for the first time they
could be nicely localized within the peripheral wall: they were found
closely beneath the primary peripheral wall and clearly within the dark
lower "secondary" layer of the peripheral wall formed during the
action of penicillin.
Consequently, in nondividing staphylococci, murosomes can hardly be
considered to exist a priori in every peripheral cell wall; rather,
these wall organelles must always be formed de novo for every new cross
wall formation. For this, murosomes are apparently placed beneath the
primary cell wall together with a rather thick layer of newly formed
secondary wall material. The possibility cannot be excluded, however,
that the murosomes are placed beneath both these layers of the
peripheral cell wall and are capable of penetrating, in a separate
step, into the secondary layer (see Fig. 19b and c).
The location of the murosomes was also monitored during the subsequent
stages of early cross wall formation (Fig. 6d and e), indicating that
the two murosomes are in some way also involved in the process of cross
wall differentiation into three parallel layers (Fig. 4a; see also Fig.
3 and 7).
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FIG. 7.
Cross-wall morphogenesis. This schematic illustration is
intended to tentatively represent the involvement of murosomes in
morphogenetic processes during neoformation of a staphylococcal cross
wall. Becoming acquainted with these morphogenetic steps is an
essential prerequisite for understanding the last minutes in the life
of a staphylococcus under penicillin (see Fig. 21 and 22). (A) Site for
cross-wall neoformation. The murosomes (MuS) are anchored within the
secondary wall (scW) directly above the site of the future cross wall
initiation. CM, cytoplasmic membrane; MWI, membrane-wall interlayer;
prW, primary wall. Reference figures, Fig. 5a and 6c. (B) Initiation of
cross wall morphogenesis. For initiation of a new cross wall, the
murosomes induce centripetally directed lytic cutting processes
considered to separate the secondary wall into three parts: a central
sector and two lateral ones. Sometimes the vesicular murosomes show
tail-like tubular extensions which, probably, are involved in this
cutting process. Reference figure, Fig. 5d to g. (C) Onset of cross
wall morphogenesis. The reason for the cutting processes within the
secondary wall seems to become evident: the central sector of the
sectioned secondary wall starts to form the so-called transitory part
of the cross wall, while the two lateral sectors initiate the formation
of the so-called permanent parts of the cross wall (see Fig. 12B).
Reference figure, Fig. 6d to e. (D) Initiation of the splitting system.
No reliable data are available about the genesis of the splitting
system (Sp). It is speculated that the splitting system stems from the
membrane-wall interlayer of the cytoplasmic membrane. Reference figure,
Fig. 2c and d.
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The stringent positioning of the murosomes certainly requires a highly
sophisticated "anchoring" procedure at the peripheral wall.
However, it is far from clear whether the apposition of secondary wall
material is the intrinsic mechanism by which the staphylococci are
capable of placing the murosomes at the necessary 90° angle to the
preceding division plane (Fig. 2b). Since the ftsZ-ftsA
genes are part of the cell division gene cluster of staphylococci
(109) one could seek to determine if they are involved in
this alteration of division planes (7). Recent findings have
shown that FtsZ can form tubular structures (83); in this regard one should consider whether there exists any relation between these tubular structures and the concentrically arranged tubuli of the
splitting systems of the cross wall (Fig. 4b).
In this connection, the reactions of staphylococci to treatment with
the bacteriostatic agent trimethoprim are especially interesting
(100). This drug is known to inhibit the growth and synthesis of secondary wall material which, after treatment with other
bacteriostatic agents such as chloramphenicol, is normally deposited as
very thick layers beneath the primary peripheral wall (see Fig. 16a).
Since during trimethoprim-mediated growth inhibition there was no such
secondary wall material beneath the primary cell wall, the murosomes
were found to be deposited between the peripheral wall and an
invagination of the cytoplasmic membrane without any detectable
association with secondary wall material; this fact was shown
especially in cells at the onset of cellular disintegration when the
region of the cytoplasmic membrane was better exposed (Fig. 6f and g).
This was the first demonstration of the formation of "free
murosomes," i.e., of murosomes not embedded in wall material (see
also Fig. 19b). These findings suggest that the apposition of new
secondary wall material below the primary peripheral wall cannot be
made responsible for the localization of the murosomes at the correct
site for inducing new cross wall initiations in the next division plane.
It was quite remarkable that in the presence of trimethoprim the
staphylococci and their murosomes were always grossly inflated but the
murosomes were located rather correctly at an angle of 90° to the
first division plane (Fig. 6f). Furthermore, a peculiar surface of the
murosomal envelope was revealed with trimethoprim, since its surface
was, for the first time, free from masking wall material. These
murosomes were shown to be covered with characteristically arranged
dark particles (Fig. 6h), the array of which could not be
differentiated from that of the typical surface of the so-called membrane-wall interlayer (43). This membrane-wall interlayer is regularly located directly beneath the peripheral cell wall (Fig. 6h
and i); it is known to cover the outer surface of the cytoplasmic
membrane of staphylococci. Earlier high-resolution freeze-etching of
this membrane-wall interlayer revealed the very peculiar, hexagonal
array of such particles, exhibiting center-to-center spacings of
approximately 7 nm (Fig. 6i).
Tentative identification of some material from the membrane-wall
interlayer located on the murosomal envelope suggests that the
possibility cannot be excluded that the cytoplasmic membrane and its
membrane-wall interlayer are in some way involved in manipulating the
position of the murosomes to the correct site for successful initiation
of subsequent cross wall formation. Questions concerning the possible
origin of the murosomes from the cytoplasmic membrane can now be
discussed as well. The surface layer of the murosomes, covered with
particles, indicates that the murosomes cannot be created simply by
invagination of the cytoplasmic membrane, since in such case the
membrane-wall interlayer would always be found inside the vesicles.
These murosomes must be formed in an evagination process, either by
evagination of the cytoplasmic membrane together with its membrane-wall
interlayer or by evagination of the membrane-wall interlayer alone. A
highly speculative schematic proposal has been sketched (Fig.
8) to elucidate the possible formation of the murosomes via local invagination of the cytoplasmic membrane, followed by murosome morphogenesis via evagination of the membrane-wall interlayer; it remains a matter of speculation whether, during an
additional step, the murosomes are capable of penetrating into the
secondary wall (Fig. 8, B-2 and C) or whether they evaginate synchronously with the synthesis of the secondary wall (Fig. 8, B and
C).
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FIG. 8.
Formation and positioning of the murosomes. A highly
speculative attempt to reconstruct the formation and localization of
staphylococcal murosomes, which are derivatives of the cytoplasmic
membrane. (A) The primary wall. A part of the primary wall (prW) is
depicted. (B-1) Formation of the secondary wall. By apposition growth
the secondary wall (scW) is placed beneath the primary wall. The
cytoplasmic membrane (CM) is indicated beneath the primary wall.
Sandwiched between the wall and the membrane is the so-called
membrane-wall interlayer (MWI). (B and B-2) Murosome morphogenesis. The
murosomes (MuS) seem to originate from a local invagination of the
cytoplasmic membrane followed by evagination of the membrane-wall
interlayer (B and B-2). It is not clear, however, whether the secondary
wall is formed before genesis of the murosomes (B-1 to B-2) or whether
it is synthesized only after the murosomes are formed (B to C).
Moreover, it cannot be excluded that murosomes and secondary wall
originate synchronously. The surface of the murosomes is covered with
particles which seem to originate from the membrane-wall interlayer.
Reference figure, Fig. 6g and h. (C) Positioning of the murosomes. At
this stage the murosomes are found to be anchored beneath the primary
wall within the secondary wall material. The murosomes are always
considered to be placed directly above the site where the next cross
wall formation will be initiated. If murosome positioning takes place
only after formation of the secondary wall (B-1 to B-2) the murosomes
must be assumed to be capable of penetrating into the secondary wall
(B-2 to C). Reference figure, Fig. 6c.
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However, judging from the contrast evident by electron microscopy, in
some cases the envelope of the transparent murosomes of control cells
was covered not only with particles but also with some other material
which looked like a rather thin but compact layer of wall material and
could be clearly differentiated from the wall material in which the
murosomes were embedded (Fig. 9a). Furthermore, some antibiotic-induced reactions of this surface layer of
the murosomes were also typical of newly formed wall material. It was
regularly thickened under chloramphenicol (Fig. 9b); such wall
thickening is highly characteristic of the action of this drug (see
Fig. 16a). In the presence of penicillin this compact layer changed to
a rather fibrillar appearance (Fig. 9c), which is well known for wall
material formed in the presence of this antibiotic (see Fig. 14a).
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FIG. 9.
Thin sections of staphylococcal cells. (a) In the
presence of sucrose a ring-like structure surrounds the murosome (MuS).
(b) In the presence of chloramphenicol (3 µg/ml) the murosome (MuS)
appears to be enveloped by a rather thick layer of wall material. (c)
In the presence of penicillin (0.1 µg/ml) the murosome is enlarged
and its surrounding layer is of rather fibrillar appearance (reproduced
with permission from reference 38). (d) During
treatment with penicillin (0.05 µg/ml) the compact cross wall has
been converted into fibrillar wall material seemingly arranged in an
arc-shaped configuration (reproduced with permission from reference
42). (e) Simultaneous treatment of staphylococci
with chloramphenicol (20 µg/ml) and penicillin (0.1 µg/ml) has
resulted in the formation of an extremely thick cross wall exhibiting a
layered architecture. (f) Under penicillin (0.1 µg/ml) cross wall
material may be assembled even at an extension of the cytoplasmic
membrane (CM), seemingly without any contact with preexisting wall
material. (g) Staphylococci having lost their splitting system during
penicillin treatment restore this system during growth in drug-free
medium (arrows) (reproduced with permission from reference
38).
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These data indicate that the murosomes, besides their lytic capacity,
can also serve, at least to a limited extent, as a special site for the
assembly of new cell wall material. The significance of such restricted
wall assembly effect is still unknown.
At any effect, the morphogenetic importance of the extraplasmatic
murosomes is indicated (i) by their sophisticated anchoring process
(Fig. 6c and f to h), (ii) by their ability to perform cutting
procedures for the initiation of cell division (Fig. 5d to g), (iii) by
their involvement in the differentiation of the three-layered cross
wall (Fig. 6d and e), and (iv) by their capability of assembling at
their surfaces at least some new wall material (Fig. 9a to c). The
subsequent induction of these processes, taking place within the
secondary wall material, is an important prerequisite for the
successful initiation and neoformation of the next cross wall at the
correct site and for the accomplishment of the subsequent cell
division which has to implement the regular alternation of the division
planes. All data available so far indicate that the murosome-induced
morphogenesis of the staphylococcal cross wall takes place as
tentatively suggested in the schematic drawing shown in Fig. 7.
These findings on cross wall morphogenesis, as summarized in the
schematic drawings in Fig. 7 and 8, are important prerequisites for
understanding the events which can be observed only minutes before
penicillin-induced death (cf. Fig. 21, B3 and Fig. 22A to D).
The lytic capacity of the murosomes is not restricted to cross-wall
morphogenesis but is also involved in two steps of lytic cell
separation: in the punching of pores into the peripheral wall (Fig.
10a and b) and in the disintegration of
transitory cross wall material (see "The lytic type of cell
separation").
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FIG. 10.
Staphylococcal cells after freeze fracturing (a to c)
and thin sectioning (d to g). (a) Cell separation starts with a row of
minute wall perforations (pores [arrows]) on the surface of the
peripheral wall above the completed cross wall (reproduced with
permission from reference 41). (b) After
deactivation of autolytic wall hydrolases by chloramphenicol (20 µg/ml) and subsequent reactivation of these enzymes by treatment with
lysozyme (10 µg/ml), two parallel rows of pores can be detected on
the surface of the peripheral wall (arrowheads) (reproduced with
permission from reference 50). (c) After
deactivation and subsequent reactivation of autolytic wall enzymes a
row of blebs (arrows) is located on the cell surface, indicating the
release of murosomes into the medium (reproduced with permission from
reference 50). (d) After chloramphenicol-mediated
deactivation and lysozyme-induced reactivation of autolytic wall
enzymes the release of murosomes (MuS) leaves behind pore-like cavities
in the peripheral cell wall (stars) (reproduced with permission from
reference 50). (e to g) The peripheral area of the
completed cross wall is shown after deactivation and subsequent
reactivation of autolytic wall enzymes. An attempt to reconstruct the
subsequent steps of murosome-mediated lytic perforation of the
peripheral cell wall during which the murosomes seem to disintegrate is
shown. (e) A murosome (arrowhead), still consisting of an envelope and
a core, appears to be rather well preserved (reproduced with permission
from reference 54). (f) The murosome (arrowhead)
shows the first signs of swelling and core disintegration (reproduced
with permission from reference 54). (g) The murosome
(arrowhead), having just perforated the peripheral wall, appears only
as an undifferentiated vesicular structure (reproduced with permission
from reference 54).
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However, no reliable experimental data are so far available concerning
the question of why murosomes perform certain vectorial lytic wall
processes only at defined stages of the cell cycle while at others they
are inactive ("resting").
Speculations about other lytically active vesicles of the
staphylococci, the so-called mesosomes, could be helpful in elucidating this problem. For mesosomes, which are also derived from the
membrane-wall interlayer, it was assumed (39) that the
autolytic wall enzymes of their vesicles (27) are regulated
by the charge of their neighboring LTAs (28). Any
transformation of their flat, "collapsed" vesicles to ball-shaped
structures would, during bulging, result in an enlargement of the outer
surface layer of the vesicle, which, in turn, would inevitably reduce
the number of LTA molecules per square nanometer and hence enlarge the
distance between the regulating LTA molecules and the regulated
autolytic wall enzymes. The resulting reduction of surface charge
density via the "blowing up" of flat vesicles could be sufficient
to transform lytically inactive vesicles into lytically active ones,
capable of attacking the staphylococcal cell wall. If such
considerations are applied to murosomes, the observation of flat,
collapsed murosomes (Fig. 5c) and ball-shaped vesicular ones (Fig. 5a)
could likewise reflect the existence of different stages of murosome activation.
The close similarities in structure, function, and genesis between
murosomes and mesosomes have led to speculations that mesosomes must be
considered as being nothing but enlarged and multiplied murosomes
induced by external factors like sucrose-mediated compression (39,
51).
Perturbations of cross wall formation.
After treatment
with penicillin, which always affects the PBPs located at the outer
surface of the cytoplasmic membrane and in the splitting system
(103), the first detectable morphological effect was the
cessation of the formation of the splitting system (47).
This observation supported the conclusion that penicillin is only
capable of inducing structural variations in growing parts of the
staphylococcal cell wall, without affecting nongrowing cell wall
regions or any part of the cell wall formed before the action of the
drug (see " `Hidden death' at high penicillin concentrations"). Therefore, perturbation of cross wall formation by penicillin proved to
be an effective tool for analyzing the structure and function of its
different components. Especially after application of very low,
nonfatal concentrations of penicillin the cross wall always revealed
its composition of fibrillar components (Fig. 9d), which was never
detected in untreated staphylococci (42). The
penicillin-induced impairment of the transpeptidation reactions necessary to cross-link the peptidoglycan strands for building up a
compact cross wall (for an overview, see reference
78) is assumed to have led to the exposure of these
fibrillar cross wall components. Remarkably, these fibrillar components
often were not randomly distributed but occurred in a seemingly
arc-shaped arrangement (Fig. 9d). Similar arrangements of fibrillar
components have been observed in several eukaryotic organisms, and they
were all interpreted as being the result of a plywood-like
superposition of layers containing more or less linearly arranged
fibrils, which only optically gives the impression of arc-shaped
structures (14, 82). Such plywood-like arrangement of
stacked macromolecular layers has been postulated to also represent the
macromolecular architecture of the staphylococcal cell wall (75,
76).
When penicillin and chloramphenicol acted simultaneously, the formation
of extremely thick cross walls was induced in which, apparently, one
layer after another of wall material was laid down, resulting in a pile
of such wall material (Fig. 9e). Such observations indicated that
layered arrangements of wall material are in fact feasible. However,
the characteristic chloramphenicol-induced thickening of the peripheral
wall (see Fig. 16a) was prevented by this combination of penicillin and
chloramphenicol (73).
Prolonged treatment with penicillin resulted in rather gross defects of
the nascent cross walls (85, 98). Sometimes, assembly of
cross wall material took place at an extension of the cytoplasmic membrane seemingly without any contact with the preexisting wall material (Fig. 9f). These data suggest that the assembly of cross wall
material will take place not only via apposition of new strands of
peptidoglycan at preexistent wall material but may start independently at the surface of certain sites of the cytoplasmic membrane
(38).
It is interesting that penicillin-induced distortions of the cross wall
as well as the disappearance of the splitting system did not persist
when staphylococci recovered after penicillin treatment in drug-free
medium (38). Under these conditions the reappearance of the
splitting system in the newly built wall material always started at the
tips of the distorted cross walls (Fig. 9g), and cross walls of normal
width were formed concomitantly.
These findings indicate that the splitting system should not be
considered exclusively as a tool for cell separation (see "An
alternative, mechanical type of cell separation using the splitting
system of the cross wall") as it may also have a function in
maintaining the highly sophisticated compact architecture of the cross wall.
Cell Separation in Staphylococci
Cell separation of fully divided bacteria is an important step in
the dissemination of daughter cells in an infection. The process of
cell separation in staphylococci seems to be unique within the
bacterial kingdom. Staphylococci developed a system of highly
sophisticated mechanisms to guarantee successful cell separation even
under adverse conditions. Depending on the growth conditions, they can
use either of two different mechanisms to separate the daughter cells
after completion of the cross wall, i.e., a lytic or a mechanical
mechanism (54).
A schematic drawing of the components involved in these mechanisms of
cell separation contributes to a better understanding of these
processes which are essential for growth and multiplication (see Fig.
12).
Initiation of cell separation via murosomes.
Earlier
observations (41) have shown that cell separation in
staphylococci always starts with the punching of a row of 18 to 24 minute wall perforations (pores) into the peripheral cell wall (Fig.
10a) above the completed cross wall. This is, apparently, the result of
the lytic activity of the murosomes of the cell wall (54).
Since calculations have suggested that cell separation in rapidly
growing staphylococci takes place within a few minutes (54),
the punching of pores will last, probably, less than 1 min. This is why
for a more detailed analysis of the mechanism of pore formation this
rapidly passing initial step of cell separation had to be slowed down.
This was accomplished by applying chloramphenicol, which is capable of
reversibly deactivating autolytic wall processes without killing the
cells (45, 50, 137). After a subsequent very slow
reactivation of the wall autolysins by cationic proteins, the process
of cell separation could be extended up to several hours, which made it
easy to follow the sequence of pore formation.
Under such specific growth conditions the peripheral pores proved, at
the very beginning of cell separation, not to be arranged in a single
row but in two parallel ones (Fig. 10b). The single row (Fig. 10a) must
be considered to be already the result of so-called pore fusion
processes (54) in which two rather small neighboring pores
are torn apart and form one larger pore. After formation of the
peripheral pores these wall perforations were extended and eventually
fused with each other, giving rise to rather large openings in the
peripheral wall. The proceeding of such opening processes, which may be
compared with the separation of two stamps along their perforation line
(see Fig. 12B), is apparently the very beginning of cell separation
(54).
A pair of circumferential sets of pores is found not only in
staphylococci but also in some oscillatoriacean cyanobacteria (33,
56, 79).
During pore formation, regularly arranged rows of blebs on the cell
surface were found to indicate a process of murosome protrusion into
the growth medium after pore punching had taken place (Fig. 10c). Such
release of murosomes after pore formation leaves behind characteristic
spherical cavities in the peripheral cell wall, as seen in thin
sections (Fig. 10d); since we are dealing here with two parallel rows
of pores, the resulting cavities are not located directly above the
cross walls but are laterally displaced.
Interestingly, the inhibition of autolytic wall processes by
chloramphenicol and their subsequent reactivation resulted in the best
preserved murosomes hitherto demonstrated by thin sectioning; often the
murosomes, 30 to 40 nm in diameter, appeared to be covered with a sort
of envelope and had a distinct core (Fig. 10e).
During perforation of the peripheral wall the murosomes often showed
signs of swelling and disintegration of their inner core (Fig. 10f and
g), indicating that we are dealing here with rather short-lived
organelles. Probably, this progressive disintegration is the very
reason why it has not yet been possible to isolate these minute wall
organelles. This tendency of self-disintegration is, probably, also the
reason why in thin sections of untreated staphylococci murosomes often
appear only as empty holes within the peripheral cell wall (Fig. 2e and
5a, d and e). This effect was even more distinct in staphylococci
growing under the influence of lytic concentrations of penicillin
(48, 50) where murosomes frequently could be detected only
as transparent lytic sites within the wall material (see Fig. 14d and e).
Wall autolysins, apparently associated with such pores, were recently
found to form a circumferential double ring on the cell surface above
the cross wall (146). These murosome-associated wall
autolysins have since been identified as
endo--N-acetylglucosaminidase and
N-acetylmuramyl-L-alanine amidase
(123).
The lytic type of cell separation.
During rapid growth
staphylococci use the method of lytic cell separation in which they
sacrifice specific central parts of their own cross wall (Fig.
11a and b). In this case the murosomes have been shown not only to perforate the peripheral cell wall via
centrifugal lytic processes but also to attack some central parts of
their cross wall proper via centripetally directed lytic actions; these
different steps of lytic cell separation have been described in more
detail recently (54). It has been concluded that the lytic
processes always start from the murosomal sites and proceed radially to
the center of the cell (54). Such bifunctional action of the
murosomes during this type of cell separation revealed that, in
staphylococci, there are both transitory and permanent parts of the
cross wall. To quantify the amount of cross wall material lost during
cell separation, the tension of the elastic cell wall had to be
reduced. This was achieved by compensating the cell turgor via
treatment of unfixed cells with 3 M sucrose. In this way it was shown
that during lytic cell separation central cross wall parts, 30 to 40 nm
thick, were lost (54), constituting about one third of the
total cross wall material (Fig. 11a and b), and which also included the
7- to 10-nm thick concentrically arranged rings of the splitting system
(Fig. 12A). These transitory cross wall
parts must be considered as being auxiliary structures exclusively
designated to be digested during cell separation (54). A
schematic drawing (Fig. 12B) depicts the onset of lytic cell separation.
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FIG. 11.
Freeze fracturing (a to d) and thin sectioning of
staphylococcal cells (e) and B. subtilis (f). (a) After
compensating the tension of the elastic cell wall by suspending unfixed
cells in 3 M sucrose (a and b), a tripartite architecture of the cross
wall was revealed (arrowheads). In the middle of the cross wall the
transitory part is located (reproduced with permission from reference
54). (b) During lytic cell separation the central,
transitory part of the cross wall is disintegrated (arrowhead)
(reproduced with permission from reference 54). (c)
After deactivation and subsequent reactivation of autolytic wall
enzymes, spoke-like intruding canals or separation scars (stars) appear
on the exposed cross wall surface after cell separation. (cW, cross
wall, pW, peripheral wall, MuS, murosome) (reproduced with permission
from reference 54). (d) A cell recovering from
chloramphenicol treatment reveals spoke-like structures (arrows) on the
just-exposed surface of the daughter cell during cell separation. (e)
After deactivation and subsequent reactivation of autolytic wall
enzymes, centripetally directed lytic wall processes (arrowheads) have
left behind a lysis-resistant central part of the cross wall
(reproduced with permission from reference 54). (f)
After deactivation and subsequent reactivation of autolytic wall
enzymes, central parts of the cross wall in a B. subtilis
cell are disintegrated during cell separation.
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FIG. 12.
Components of staphylococcal cross walls. These
sketches (modified from reference 54) give
preliminary information about the cross wall components involved in
cell division and in the different types of cell separation of
staphylococci. (A) Situation during initiation of cell separation.
Illustrated is a divided staphylococcus with a newly completed cross
wall before cell separation liberates the two daughter cells; however,
in order to look inside the cross wall with its divers components, the
right daughter cell is depicted separately, at some distance from its
normal location. Cell separation is just being initiated by the
centrifugally directed lytic activity of the murosomes (MuS) which
punch two rows of pores (po) into the peripheral cell wall. In slowly
growing staphylococci cell separation takes place along the
concentrically arranged rings of the splitting system (Sp) which is
synthesized during cross wall formation; in rapidly growing
staphylococci cell separation takes place along the spoke-shaped canals
(spo) which originate only after completion of the cross wall by the
centripetally directed lytic activity of the murosomes. The cross wall
material located between the two rows of pores including the splitting
system is only destined for cell separation and will be disintegrated;
this material can only be considered as being transitory parts of the
staphylococcal cross wall. Reference figures, Fig. 4b and 11d. (B)
Situation after the onset of cell separation. Illustrated are those
parts of a completed cross wall which are located directly beneath the
peripheral cell wall (pW). In a first lytic step the murosomes (MuS)
have punched, via their centrifugal lytic activity, two circumferential
rows of pores (po) into the peripheral wall (upward arrow, left side).
These pores in the peripheral wall are then torn apart along the
perforation line (right row). In a second lytic step the murosomes
attack central parts of the cross wall via centripetally directed lytic
actions (downward arrow, left side), resulting in the formation of
spoke-shaped canals (spo). Between the presumptive cell walls of the
future daughter cells (dW) and the peripheral wall of the mother cell
(pW) is the location of the so-called stripping system (Str) which is
involved in cell wall turnover. Reference figures, Fig. 10a and b and e
to g and Fig. 11c to e.
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The muralytic enzymes involved in this separation process are,
apparently, the same enzymes that are capable of punching the pores
into the peripheral cell wall for initiating cell separation, i.e., two amidases, the
endo--N-acetylglucosaminidase and the N-acetylmuramyl-L-alanine amidase
(122), which were previously shown to be capable of
dispersing cluster-forming mutants of staphylococci (20)
(see "Inhibition of cell separation results in the formation of
pseudomulticellular staphylococci").
Experimentally induced lytic cell separation aside from the
splitting system.
For a more detailed analysis, the rapid process
of lytic cell separation also had to be slowed down via reversal
deactivation of wall autolysins by limited chloramphenicol treatment,
followed by a slow reactivation of lytic wall processes by cationic
proteins ("see Initiation of cell separation via murosomes"). The
utilization of this technique is considered to be capable of
demonstrating the original pattern of distribution of the bulk of
staphylococcal wall autolysins (50). Furthermore, the
chloramphenicol-induced thickening of the staphylococcal wall (40,
42) (see "Wall thickening via underlayering processes") has
proven to be of great advantage for ultrastructural analysis.
After application of this slowing-down technique, centripetally
intruding spoke-like canals (Fig. 11c) were demonstrated on the surface
of the daughter cells (54), which canals to a certain extent
resemble the so-called separation scars on the cell surface of yeasts
(133).
Apparently, the spoke-like canals of the staphylococci are the result
of the reactivated lytic activity of wall autolysins starting from the
site of the peripheral murosomes. Even more important was the
observation that very differentiated spoke-like structures, resembling
chrysanthemum flowers (Fig. 12A), were also found in some staphylococci
recovering from chloramphenicol treatment (Fig. 11d). Below it will be
seen that the formation of spoke-like canals in the cross wall could
also be induced by treatment with penicillin (see Fig. 14e).
Demonstration of these radially orientated canals in the cross wall
under different experimental conditions is of considerable interest
since it confirms the conclusion that in control cells the muralytic
processes during lytic cell separation, as mentioned in the preceding
section, also start from the murosomal sites and proceed radially into
the center of the cell. Furthermore, these findings suggest that the
canals cannot be considered as being artificial conformations created
by the experimental conditions.
So far there has been no convincing explanation for the preservation of
the spoke-shaped tracks under some experimental conditions. They have
only been found in staphylococci treated with chloramphenicol or
penicillin, and it is suggested that they are rapidly dissolved after
the centripetally directed muralytic actions of the murosomes, probably
by the activity of the so-called stripping system (see "Wall
thickening via underlayering processes" and Fig. 12B). Since after
treatment with chloramphenicol all autolytic processes are in some way
retarded, one could also speculate that the final cleaning of the
surface of the daughter cells, i.e., the complete disintegration of all
parts of the cross wall which are to be dissolved, takes more time than
in control cells.
At least the induced lytic processes in the cross wall, proceeding
centripetally from the peripheral pairs of murosomal sites, at first
hardly attack the central region of the cross wall which contains the
splitting system; sometimes these processes spared even the entire
central part of the cross wall (Fig. 11e).
It should be pointed out that the spared part of the cross wall holds
about 30% of the entire cross wall material and also comprises the
splitting system; it is, therefore, much thicker than the splitting
system proper (only 7 to 10 nm in width) (103). Furthermore,
this central part of the cross wall reaches up to the cell periphery,
while the splitting system itself is known to be restricted to the
slit-like container of the cross wall which extends only to the inner
surface of the peripheral wall (Fig. 3). A schematic drawing shows the
limited disintegration of transitory parts of the staphylococcal cross
wall during this type of cell separation (Fig. 12B).
The fact that the procedure of reactivating autolytic wall enzymes was
not capable of inducing lytic processes within the splitting system
argues for the assumption that the splitting system contains hardly any
autolytic wall enzymes and is normally not involved in this type of
lytic cell separation. Hence, one could presume that induced lytic cell
separation is mainly the result of the two vectorial lytic activities
of the murosomes directed centrifugally and centripetally, supplemented
by the autolysins of the stripping system.
A similar process of cell separation, in which certain parts of the
cross wall are sacrificed, was also observed in cells of Bacillus
subtilis (Fig. 11f), indicating that disintegration of substantial
parts of the cross wall during cell separation also takes place in
another gram-positive bacterium.
An alternative, mechanical type of cell separation using
the splitting system of the cross wall.
During slow growth, for
instance in the stationary phase of growth, cell separation again
starts with the activation of the centrifugally directed lytic capacity
of the murosomes, resulting in the punching of pores into the
peripheral cell wall as described above (see "Initiation of cell
separation via murosomes"). However, no centripetal lytic processes
start out from the peripheral pores, since under these conditions
staphylococci utilize mainly the splitting system for cell separation.
This method is considered as an alternative, more or less nonlytic,
technique for completing the fission process without sacrificing
greater parts of the cross wall.
For most of the investigated staphylococcal strains, during slow growth
the thin (7 to 10 nm) splitting system of the cross wall disappears in
the course of cell separation. One normally observes that after this
type of cell separation, running along the splitting system (Fig.
13a), not even traces of their
concentric tubuli can be detected on the just-exposed surface of the
nascent daughter cells (Fig. 13b) (3). Since no lytic
activity could be demonstrated within the splitting system, the
immediate disappearance of its tubuli at the onset of cell separation
is not yet completely understood. One could speculate that in this case
staphylococci utilize the preformed longitudinal slit in the cross wall
which contains the tubuli of the splitting system (Fig. 3D) for cell separation and that the tiny tubuli are destroyed or lost during this
event.
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FIG. 13.
Thin sections (a and g) and freeze fractures (b to f)
of staphylococcal cells. (a) Cell separation in untreated cells along
the small layer of the splitting system without detectable loss of
cross wall material. (b) During the separation of untreated cells not
even remnants of the concentrically arranged tubuli of the splitting
system can be detected on the just-exposed cross wall surfaces of the
daughter cells. Only the clefts (arrowheads) are preserved which mark
the site of the initial cutting through of the peripheral wall
(reproduced with permission from reference 54). (c)
After growth in the presence of chloramphenicol (20 µg/ml) and
subsequent regeneration in drug-free medium, the concentrically
arranged tubuli of the splitting system are preserved on the
just-exposed surfaces of the daughter cells (reproduced with permission
from reference 44). (d) This untreated cell of
S. aureus SA 113 reveals the concentrically arranged tubuli
of the splitting system on the cross wall surfaces of both daughter
cells during cell separation. (e) After growth in the presence of
chloramphenicol (20 µg/ml) and subsequent regeneration in drug-free
medium, the next division plane is already initiated beneath the center
of the still-preserved concentrically arranged rings of the splitting
system (arrowheads) (reproduced with permission from reference
44). (f) In the presence of penicillin (0.1 µg/ml), the murosome-mediated punching of holes into the peripheral
wall for cell separation starts in a zigzag-like manner (stars),
resulting in the formation of two parallel rows of circumferential
pores (reproduced with permission from reference
38). (g) In spite of growth in the presence of
penicillin, this murosome just released from the cell appears to be
rather well preserved.
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However, after blocking the muralytic activity with chloramphenicol,
which leads to a drastically reduced wall turnover without killing the
cells, and after subsequent regeneration of the cells during growth in
any normal medium, remnants of the concentrically arranged tubular
rings were preserved and nicely exposed on the surface of the daughter
cells (42, 44) (Fig. 13c). During all stages of this fission
process, when the exposed cross walls started bulging, more or less
complementary images of the concentric rings were on the surface of the
cross wall of both daughter cells detected (Fig. 13c). These findings
have proved that in this case the process of cell separation actually
takes place exactly within the middle of the splitting system, i.e.,
the concentrically arranged tubular rings only 7 to 10 nm wide must be
considered to represent the very splitting plane of this type of cell
separation. Furthermore, unlike in S. aureus, in S. epidermidis such concentric circular structures are already
visible on the surface of control cells (4). The splitting
system of a certain mutant strain of S. aureus,
characterized by a low turnover rate, was also shown to persist during
cell separation (Fig. 13d, strain SA 113) (54, 65). All
these observations have shown that cell separation per se will not
disintegrate the tubuli of the splitting system.
In discussing the question of why during slow growth the splitting
system is sometimes disintegrated but is preserved in other cases, a
look at the fate of preserved splitting systems should prove to be
helpful. After the first cell separation the next division plane in the
more or less ball-shaped daughter cells is initiated directly beneath
the center of the concentric ring system (Fig. 13e), and the
concentrically arranged rings are still preserved even at this late
division stage; only much later were they disintegrated via turnover
processes during the next generation time. Such turnover processes,
being the result of a so-called stripping system, were capable of
disintegrating even rather thick layers of wall material (see "Wall
thickening via underlayering processes"). It is conceivable,
therefore, that the wall enzymes of this stripping system that are
normally competent for wall turnover could be responsible for the
disintegration of the tubular structures of the splitting system during
cell separation of slowly growing staphylococci. However, experimental
data which might 
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