JOURNAL OF BACTERIOLOGY, Mar. 1976, p. 1139-1147 Vol. 125, No.

3
Copyright 0 1976 American Society for Microbiology Printed in U.SA.

Layered Distribution, According to Age, Within the Cell Wall

of Bacillus subtilis
H. M. POOLEY'
National Institute for Medical Research, Mill Hill, London NW7 1AA, England
Received for publication 22 September 1975

When soluble autolytic activity was added to growing cultures of a mutant

possessing a reduced rate of cell wall turnover, there was a delay of more than
one generation before solubilization of new cell wall began, in contrast to the
immediate increase in the rate of solubilization of old cell wall. A similar delay

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was found before turnover of new cell wall occurred in the parent, in agreement
with a previous report (Mauck et al., 1971). When sodium lauryl sulfate-
inactivated cell walls were prepared, the great bulk of the wall formed a
uniformly susceptible substrate to added autolytic activity. The immediate
solubilization of new wall eliminates insusceptibility to autolytic enzyme as an
explanation for the failure to be turned over. There were, however, major
differences in the rate of solubilization of wall of different ages. During solubili-
zation of the initial 30% of the cell wall preparation, wall two generations old
was solubilized at least seven times faster than wall one-half a generation old.
This result is interpreted in terms of differences in accessibility. The cell wall is
seen as consisting of a series of layers, the age of which increases with the
distance from the membrane, such that wall newly synthesized on the mem-
brane passes out through the thickness of the cell wall layer during subsequent
growth and only becomes susceptible to turnover as it reaches the outer surface,
largely in the form of a layer, more than one generation after incorporation.
I have presented evidence (13) that uniformly dophilus (1). The reasons for the failure of
labeled wall occupies an exponentially increas- newly incorporated mucopeptide to turnover in
ing surface area for up to two generations after rod-shaped organisms have not been estab-
a chase and forms a shallow outer layer, from lished. I hoped to resolve this question in terms
which all turnover takes place and which over- of the geometry of arrangement of wall of differ-
lies newer wall. In the present study I have ent ages within the cell wall layer. Accord-
sought to provide support for such an arrange- ingly, I have employed isolated cell walls
ment in two ways. (i) It has been shown (13) treated with sodium lauryl sulfate (SLS) to
that the rate of solubilization of the cell wall of inactivate associated lytic enzymes to establish
a mutant possessing a reduced rate of turnover that the turnover pattern for new wall is not
can be greatly increased by the addition of auto- due to differences in substrate susceptibility of
lytic activity to growing cultures. In the pres- wall of different ages. In addition, by isolating
ent study, the kinetics of solubilization of pulse- walls at different intervals after chasing a
labeled wall by added autolytic activity have pulse-labeled culture, I hoped to observe any
been investigated. (ii) The accessibility of changes in the position of the labeled wall
newly synthesized wall to added autolytic en- within the cell wall layer, during surface
zyme has been studied in isolated cell wall growth, by looking for changes in its accessibil-
preparations. ity to added autolytic activity. The results have
A delay before solubilization of new wall by led me to an apparently novel conclusion about
autolytic activity added to growing cultures the distribution of wall of different ages within
was expected, comparable to the period of the the cell wall layer. I have discussed (13) the
exponentially increasing rate of turnover from importance of such studies in connection with
pre-existing wall (13). Indeed, delays a genera- the continuing disagreement in the literature
tion long before turnover of pulse-labeled wall (4, 5, 7, 8, 12) over the pattern of insertion of
have been reported in Bacillus subtilis and B. newly synthesized wall during surface expan-
megaterium (3, 11) and in Lactobacillus aci- sion in bacilli.
(A preliminary report of this work was given
I
Present address: Institut de Microbiologie, Universite at the January 1975 meeting of the Society For
de Lausanne, 1005 Lausanne, Switerland. General Microbiology, Brighton, England.)
1139
1140 POOLEY
MATERIALS AND METHODS
Organisms. The bacterial strains used in these
experiments were B. subtilis 168 trp-thy- and a mu-
tant showing a reduced rate of cell wall turnover,
Nil5, which was obtained from the above strain (see
[131 for details of isolation).
Media for cultural conditions. Media and cul-
tural conditions were as described (13). The growth
medium in the present study was a casein hydroly-
sate medium supplemented with inorganic salts (9)
used at one-half strength.
Incorporation of [14C]GlnAc into the cell wall
fraction and measurement of cell wall turnover.
The procedures employed for incorporation of N-
acetyl-D-[1-14C]glucosamine (['4CIGlnAc) into the
cell wall fraction and measurement of cell wall
turnover have been described (13).
Pulse-labeling the cell wall fraction. The results
of measuring incorporation of ['4C]GlnAc into the
cell wall during pulse-labeling of 0.15 generations (3
min) showed that, immediately after the cells were
removed from the labeled medium, the radioactivity
extracted within 1 min of treatment with cold 5%
trichloroacetic acid represented 12 to 14% of the total
radioactivity in the cell wall fraction. (The corre-
sponding figure for continuously labeled walls was 3
to 4%.) The total radioactivity incorporated into the
cell wall fraction that was insoluble in trichloroace-
tic acid increased by approximately 15% during the
first quarter of a generation (4 to 5 min) after re-
moval of the cells from radioactive precursor. This
latter figure is taken as the total radioactivity incor-
porated. Measuring turnover by the release of radio-
active turnover products to the medium is unambig-
uous and independent of whichever value is used for
the total radioactivity incorporated into the cell wall
fraction. Immediately after resuspending the cells
in unlabeled medium, if the cells were collected
directly on a membrane filter, a small amount of
soluble radioactive material was found to be pres-
ent, equivalent to approximately 6 to 8% of total
radioactivity incorporated. This was most probably
due to carryover from the labeled medium. The cor-
responding value for continuously labeled cultures
was 2 to 4%. The soluble radioactive material pres-
ent at zero time was subtracted from the nonfiltera-
ble radioactivity of all samples.
Preparation of SLS-inactivated cell walls. The
procedures used for obtaining broken cell prepara-
tions and for the isolation of native, autolytically
active walls have been described (13). When autolyt-
ically inactive (denatured) cell walls were required,
carrier cells, grown under the same conditions and
harvested at about the same optical density (OD),
were added to labeled cells before cell breakage. The
broken cell preparation was treated with an equal
volume of 4% (wt/vol) SLS, followed by centrifuga-
tion at room temperature for 15 min at 36,000 x g.
The pellet was resuspended in 2% SLS and heated to
55 C for 5 min before being centrifuged again. The
wall preparation was subjected to the same washing
procedures as those used for the preparation of na-
tive walls and freeze dried.
Lysis of radioactively labeled SLS-inactivated
J. BACTERIOL.
walls. The preparation of soluble cell wall autol-
ysates has been described (13). Lysis was car-
ried out in most instances as follows. SLS-inacti-
vated "4C-labeled cell walls were suspended in water
at a concentration of 10 mg/ml. A 0.6-ml portion was
added to a tube standing on ice containing cell wall
autolysate prepared from approximately 4 mg of cell
walls And potassium phosphate buffer (pH 7.0 at a
final concentration of 0.05 M) in a total volume made
up to 6.0 ml with water. After mixing, the tube was
placed in a water bath at 45 C, and the OD4 50 was
measured at intervals. To measure the solubiliza-
tion of radioactively labeled material during lysis,
300-,ul samples of the mixture were removed at in-
tervals, added to 300 pJ of 4% SLS in a Pyrex centri-
fuge tube (0.8 by 7.5 cm), and mixed. These samples
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were centrifuged at 20,000 x g for 15 min, and two
250-,Iu volumes of the supernatants were added to
glass fiber disks contained in scintillation vials. The
remaining supernatants were discarded without dis-
turbing the cell wall pellets, to which 2 ml of water
was then added, and the tubes were centrifuged
again. These supernatants were discarded, and the
pellets were suspended in 0.2 ml of water and trans-
ferred quantitatively, by several further washings,
on to glass fiber disks contained in scintillation
vials. All the disks were dried down at 60 C, and 5
ml of Biosolve-toluene scintillation fluid was added.
The 14C counting efficiency was 90%. Insoluble ra-
dioactive material and the turbidity, expressed as
the percentage of zero time readings, were plotted
against time at 45 C. To correct for a small, varia-
ble, and rapid loss of turbidity that occurred in the
first 1 or 2 min of lysis, 100% turbidity was taken as
the value obtained by extrapolation of the initial
linear part of the lysis curve to zero time.
RESULTS
Turnover of pulse-labeled and continuously
labeled cell wall in the parent strain. When
cultures of B. subtilis 168, pulse-labeled and
continuously labeled with [14C]GlnAc, were al-
lowed to continue growth in the presence of
unlabeled GlnAc, the release of radioactive
turnover products was as shown in Fig. 1. Less
than 5% of the total radioactivity in the cell
wall was released from pulse-labeled cells dur-
ing the first generation and one-half. A similar,
although shorter, lag has been reported previ-
ously for B. subtilis (11). With the expected
pattern of incorporation of 70 to 75% of the label
in mucopeptide (14), and the remainder in tei-
choic acid, this delay before release of label is
also consistent with previous results (11) show-
ing that the kinetics of turnover of both cell
wall polymers, labeled during a pulse, follow
the same pattern. No significant fraction of the
radioactivity became subject to turnover for
about 30 min after the chase. This lag period
was followed by a rapid loss of pulse-labeled cell
wall material, 30% being released within the
VOL. 125, 1976 AGE-DETERMINED LAYERS IN THE CELL WALL
Minutes
20 40 60
100
D 80
*-_
70
0 reduced rate of wall turnover, can be stimu-
r 60
O""o
FIG. 1. Turnover of pulse-labeled and continu-
ously labeled cell wall in B. subtilis. The B. subtilis
parent strain was grown in medium containing 60
JM ['4C]GlnAc (specific activity, 5 jACi/,Amol) for
more than five generations before 3.5 ml of the cul-
ture was filtered at an OD 6 75 of 020. The cells were
washed and suspended in 3.5 ml of nonradioactive
medium. A 2-ml portion ofthe resuspended cells was
then added to a flask containing 10 ml of nonradio-
active medium and growth was allowed to continue.
A 4-ml sample of a second culture, growing in me-
dium containing nonradioactive GlnAc, was filtered
at an OD of 0.21, and the cells were washed and
rapidly suspended in 3.5 ml of medium containing
10 pM ["4CJGlnAc (specific activity, 45 puCi/,umol).
After 0.15 generations (3 min), the culture was refil-
tered, washed, and suspended in 3.5 ml of unlabeled
medium, from which a 1.5-ml sample was with-
drawn and added to a flask containing 10 ml of
medium. During subsequent growth in the nonradio-
active medium, the radioactivity in the cell wall frac-
tion and that released to the medium was measured
as described (see Materials and Methods and [13D).
The radioactivity remaining in the cell wall fraction
is shown for continuously labeled (0) and for pulse-
labeled (-) cultures as a percentage of the total ra-
dioactivity incorporated. One hundred percent radio-
activity incorporated into the cell wall fraction for the
pulse-labeled culture was 9.2 x 103 dpm/ml and for
the continuously labeled culture was 1.04 x 104
dpm/ml.
1141
next one and one-half generations. In contrast,
the release of radioactivity from continuously
labeled cells proceeded without an initial lag,
being about 20% complete in the first one and
one-half generations, although the maximum
rate of turnover was not reached until after
more than one generation of growth in unla-
beled medium, in agreement with the result
found for a culture growing on a slightly differ-
ent medium (13). The nonrelease of turnover
products from pulse-labeled wall suggests that
newly synthesized wall is either insusceptible,
or inaccessible, to the action of the autolytic
activity responsible for wall turnover.
Accessibility of new and old cell wall to
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added autolytic activity in growing cultures
of the mutant Nil5. The rate ofcell wall solubi-
lization of continuously labeled cultures of
Nil5, a mutant which has been shown to have a
lated by the addition of autolytic activity to
growing cultures (13). The effect on cell wall
solubilization when pulse-labeled or continu-
ously labeled cultures of Nil5 were grown in
the presence of added autolytic activity in non-
radioactive medium is shown in Fig. 2. The
pulse-labeled cells showed a very slow loss of
radioactivity for about one and one-half genera-
tions (25 to 30 min) of growth in unlabeled
medium, even when grown in the continuous
presence of autolytic activity.
There was no difference in release of radioac-
tivity throughout this period, whether the
pulse-labeled culture was grown in the pres-
ence or absence of autolysate. This experiment
indicates that new, pulse-labeled wall did not
become accessible to externally added autolytic
activity in exponentially growing cultures for
about one and one-half generations after incor-
poration. In contrast, there was an immediate,
rapid, and constant rate of loss of radioactivity
from continuously labeled cells, in agreement
with results found in similar experiments al-
ready described (13), which indicated that old
cell wall was immediately susceptible to exter-
nally added autolytic activity. Cell lysis during
growth in the presence of added autolysate was
not responsible for the pattern of turnover ap-
parently observed (13).
Addition of autolytic activity to growing
cultures of pulse-labeled Nil5 at various times
after incorporation. A pulse-labeled culture of
Nil5 was divided into four parts and allowed to
continue growth in the presence of nonradioac-
tive GlnAc. Soluble autolysate was added to
three parts, at zero time (Fig. 3B), at 0.8 gener-
ations (16 min) (Fig. 3D), and at 2 generations
(40 min) (Fig. 3C), respectively, after removing
1142 POOLEY J. BACTZRIOL.
Minutes

r's0 \
IL ~
~ ~~~\Pulse
V' Autolysin
Al!'60 60 -
Addition
cc~~~~~~~~N
Omirt
6 40 Continuous\

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20~~~~~~
0 416min.
40ri
FIG. 2. Release of radioactively labeled turnover
products from new and old cell wall in cultures of
Nil5 growing in the presence of added autolytic en- FIG. 3. Addition of autolytic activity at various
zyme. A 3.5-ml portion ofa culture of Nil5 grown for times during the chase period of pulse-labeled cul-
more than five generations in medium containing 55 tures ofNil5: the effect on the time of appearance of
pM [t'4ClGlnAc (specific activity, 5 ACi4Amol) was pulse-label susceptibility to turnover. An 8-ml portion
filtered at an OD of 0.22 and suspended in 3.5 ml of of a culture of Nil5 growing in the presence of 60 pM
medium containing nonradioactive GlnAc. A 1-ml GInAc was filtered at an OD of 0.24, and the cells
portion ofthe resuspended cells was added to 10 ml of were suspended in 6 ml of medium containing 10 pM
medium containing soluble autolysate (1.5 mg of ['4CJGlnAc (specific activity, 83 pCi/pmol). After less
autolyzed wall/OD per ml of bacterial culture), and than 0.2 generations (3 min), the cells were refiltered,
growth was allowed to continue. A 3.5-ml portion of a washed, and suspended in 6.0 ml of medium contain-
second culture grown in the presence of55 M GInAc ing unlabeled GlnAc. Samples (1, 2 and, 2.4 ml,
was filtered at an OD of 0.21, and the cells were respectively) were removed and added to three flasks,
suspended in 3 ml of medium containing 10 pM A, B, and C, containing 10, 20, and 24 ml ofmedium,
[14C]GlnAc (specific activity, 50 jACilpmol). After 0.2 respectively. The cells in flask A served as the control
generations (3.5 min) of growth, the culture was for turnover of the culture grown in the absence of
refiltered and the cells were suspended in 2.5 ml of autolysate (0). Flask B contained 100 yd of soluble
medium containing nonradioactive GlnAc. Samples autolysate (5 mg of autolyzed wall/ml) which gave a
(1 ml) of resuspended cells were added to two flasks, final concentration of 0.65 mg of autolyzed wall/OD
each containing 10 ml of medium, only one of which per ml of culture (O). Growth of cells in flask C was
contained soluble autolysate (1.5 mg of autolyzed allowed to continue without addition for 0.8 genera-
walls/OD per ml). Growth was allowed to continue, tions (16 min), when 12 ml was withdrawn and
and 1-ml samples were removed at intervals for added to a fourth flask, D, which contained 100 pg of
measurement of the radioactivity remaining in the soluble autolysate to give a final concentration of 0.75
cell wall fraction and that released to the medium mg of wall/OD per ml (A). After two generations,
during turnover as described. The radioactivity re- 200 ji of autolysate was added to the cells remaining
maining in the cell wall, as a percentage of the total in flask C to give a final concentration of autolysate of
incorporated, is shown for pulse-labeled (A) and con- 0.70 mg of autolyzed wall/OD per ml of culture (U).
tinuously labeled (0) cells grown in the presence of The time of addition of autolysate is indicated by an
autolysate. Also shown is the release from pulse- arrow. One hundred percent radioactivity in the cell
labeled cells (0) grown without added autolysate- or wall for all cultures was £25 x 104 dpm/ml.
pulse-labeled cells. One hundred percent 14C was 1.0
x 104 dpm/ml and, for continuously labeled cells, it presence of autolytic activity. This shows that
was 9.4 x 103 dpmlml. the time at which pulse-labeled wall became
accessible to added autolytic activity was not
the cells from labeled medium. A fourth part dependent upon the time of autolysate addition.
(Fig. 3A) served as an untreated control. The The release of radioactivity from the two-gener-
pattern of release of radioactivity obtained is ation part (Fig. 3C), indicated that after two
shown in Fig. 3. The time of onset of rapid loss generations the pulse-label was largely accessi-
of radioactivity was identical in the zero-time ble to added autolytic activity, even though it
and 0.8-generation parts, although the cultures was not being turned over in the control culture
had been grown for different periods in the (Fig. 3A) of the mutant. The results for pulse-
VOL. 125, 1976
labeled cells were in contrast to those obtained
in similar experiments performed with continu-
ously labeled cells (13), where the rate of solubi-
lization of radioactivity was increased immedi-
ately after addition of soluble autolytic enzyme.
Susceptibility of new and old wall to added
autolytic activity in SLS-inactivated wall
preparations. SLS-inactivated cell walls pre-
pared from pulse-labeled and continuously la-
beled cultures of Nil5 were lysed by the addi-
tion of soluble autolysate (Fig. 4). The release of
radioactivity during lysis of pulse-labeled walls
(Fig. 4B) proceeded without any lag and at a
rate slightly faster than the overall rate of loss
AGE-DETERMINED LAYERS IN THE CELL WALL 1143
4A) showed the parallel loss of turbidity and
release of radioactivity expected of a uniformly
labeled substrate. Although the loss of turbid-
ity is registered almost entirely by the carrier
walls, the parallel loss of 14C and turbidity indi-
cates that there are no differences in suscepti-
bility between the labeled and carrier walls. In
a similar experiment (data not shown), more
than 80% of the radioactivity was solubilized at
a constant rate. If a further addition of autoly-
tic activity was made, doubling the initial con-
centration per milliliter when half of the wall
had been solubilized (Fig. 5), the rate of loss of
radioactivity doubled without a perceptible lag.

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of turbidity. The rate of loss of turbidity de-
clined markedly from linearity after loss of 60
to 70% of the initial turbidity (data not shown).
The immediate release of pulse-labeled wall
clearly indicated that new wall, immediately
after incorporation, was equally or even more
susceptible than old wall to externally added
enzyme. This result rules out the possibility
that insusceptibility to autolytic attack of the
pulse-labeled wall might be the explanation for
the failure to be turned over.
The lysis of continously labeled walls (Fig.
The final rate of release remained constant un-
til at least 90% of the wall had been solubilized.
These results indicate that the added activity
was not substrate limited during this period,
and that the great bulk of the wall formed a
uniformly susceptible substrate. This conclu-
sion is important for the interpretation of the
results described below and for the conclusions
obtained from the turnover studies (13) con-
cerning the distribution of old wall within the
cell wall layer.
Variation in susceptibility of wall of differ-

Minutes
20 40 0 20
,VV X I I I I

A. Continuous 0a B. Pulse
0o0

80- 0
0\ o *

0.P 8\.
o* 450
%6o~~~ N
14C o

0
40 \0
0

FIG. 4. Susceptibility of new and old cell wall to added autolytic activity in SLS-inactivated cell walls. A
culture of Nil5 was grown in 10 ml of medium containing 60 [14C]GlnAc to an OD of 0.23 before being
harvested by filtration. A second culture was grown in 10 ml of medium containing 60 pM [14C]GlnAc to an
OD of020 before being filtered, and the cells were suspended in 10 ml of medium containing 4 pM ['4C]GlnAc
(specific activity, 250 juCiIpmol). After 0.15 generations (3 min), the culture was harvested by filtration. An
identical amount of carrier cells (ca. 02 g, dry weight) was added to both samples of harvested cells, and SLS-
inactivated walls were prepared as described. 6.1 mg and 4.7 mg of cell wall from continuously (A) and pulse-
labeled (B) cells, respectively, were lysed by the addition of 0.2 ml of a soluble autolysate (20 mg of autolyzed
wallIml), prepared as described (13), in a total volume of 6.0 ml. The details of lysis conditions and
sampling are described in Materials and Methods. Insoluble radioactivity (0) and OD45 (-) values are shown
0

as a percentage of those at zero time plotted against time. All the values for pulse-labeled wall lysis have been
multiplied by 0.78 (i.e., 4.716.1) to obtain the rate of lysis at an equal autolysate concentration to that of the
continuously labeled cell wall lysis. One hundred percent radioactivity for (A) was 2.95 x 104 dpm/mg and
was 2.74 x 104 dpm/mg for (B).
1144 POOLEY
Minutes
0
.Q
a
C5
FIG. 5. The substrate susceptibility, during lysis,
of SLS-inactivated walls. The cell walls used were
those described in the legend to Fig. 4. The procedure
used for the preparation of a cell wall autolysate and
measurement of lysis was as described in Materials
and Methods and in the accompanying paper (13). A
120-pl portion of cell wall autolysate (10 mg of walll
ml) was added to 4.4 mg ofwalls in a total volume of
4.5 ml. A control without added autolysate contained
1.1 mg of walls in a total volume of 1 ml. The lysis
was allowed to proceed, and samples were removed at
intervals. At the time indicated (see arrow), a further
addition of autolysate (90 pd) was made to the re-
maining 3.3 ml, doubling the initial concentration of
autolysate per milliliter. Insoluble radioactivity as a
percentage of the zero time value is shown for walls
incubated with (a) and without (0) added autoly-
sate.
ent ages to autolytic attack in isolated SLS-
inactivated wall preparations. A pulse-labeled
culture of Nil5 was allowed to continue growth
in the presence of nonradioactive GlnAc. SLS-
inactivated cell walls were isolated from sam-
ples taken at various times after pulse labeling.
The lysis of the isolated walls by added autoly-
tic activity is shown in Fig. 6. The release of
radioactivity was almost parallel to loss of tur-
bidity in walls isolated immediately after incor-
poration of the pulse label (Fig. 6A); 46% of the
radioactivity was solubilized during the initial
50% drop in turbidity. In contrast, during lysis
of walls isolated one-half generation after chas-
ing (Fig. 6B), the loss of radioactivity was much
slower than the loss of turbidity during the
initial stages of lysis; barely 8% was solubilized
during the first 30% drop in turbidity. When
more than 50% of the turbidity had been lost,
the rate of release of radioactivity had in-
creased, to become faster than the overall rate
of lysis (as indicated by the rate of loss of tur-
bidity). The loss of radioactivity from walls iso-
lated from cells grown for two generations in
nonlabeled medium (Fig. 6C) was very rapid;
40% of the radioactivity was solubilized during
the initial 20% loss of turbidity.. These large
J. BACTERIOL.
differences in the rate of solubilization of wall
of different ages might be explained by a time-
dependent maturation process in vivo. How-
ever, such a hypothesis would not explain the
immediate susceptibility in vitro of new wall.
In addition, since any such process would be
arrested during wall isolation, this hypothesis
is not compatible with the uniform susceptibil-
ity shown by at least 90% of the wall in vitro
(Fig. 4A and 5). I therefore believe that the lysis
curves shown in Fig. 6 indicate that there are
major differences in accessibility to externally
added autolytic activity of cell wall fractions of
different ages. The most accessible wall was
that two generations old, followed by newly
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synthesized wall, with wall one-half a genera-
tion old being almost inaccessible to added au-
tolysin.
When SLS-inactivated walls were isolated
two generations after chasing a continuously
labeled culture, the release of radioactivity dur-
ing lysis (Fig. 7) was initially twice as fast as
the loss ofturbidity, an identical pattern to that
found for wall isolated from a pulse-labeled cul-
ture, after two generations of growth in unla-
beled medium (Fig. 60). Since all wall is appar-
ently equally susceptible, this result indicated
that wall two generations old had the same
accessibility as all older wall, having bound a
markedly higher than average concentration of
autolytic activity. If the bulk of the externally
added enzyme had become bound to the outer
surface of the isolated walls, the result would
suggest that old wall exists at higher concen-
tration on the surface of the wall layer. This is
the distribution which was proposed from the
turnover pattern of old cell wall in the intact
organism (13).
DISCUSSION
The accessibility to added autolytic activity
of wall of different ages in isolated cell wall
preparations is in good agreement with the re-
sults obtained by the addition of autolytic activ-
ity to growing cells, with one exception. This is
the accessibility, in isolated walls only, of
newly incorporated wall material. Pulse-la-
beled mucopeptide is probably incorporated at
the inner surface of the wall layer, adjacent to
the membrane. This area of the wall would be
least accessible to enzyme added to the outside
of intact growing cells, but in broken cell wall
preparations this inner surface ofthe wall layer
would be accessible to added enzyme. The pulse-
label, two generations after incorporation, is
released from isolated cell walls by added auto-
lytic activity much more rapidly than the aver-
age rate at which wall is digested, which im-
VOL. 125, 1976 AGE-DETERMINED LAYERS IN THE CELL WALL 1145
Minutes
0 20 40 0 20 40 0 20 40

10 I 00 O 'C \ '
80 00
0~ ~ ~
0

I ~~~~~0
%6o ~~0%A 0

ODQ450 %
0O0.
45045 %~~~~~~

%
0 %

40 0~~~~

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20~~~~~~~~

FIG. 6. Variation in susceptibility of cell wall material of different ages to autolytic attack in isolated SLS-
inactivated wall preparations. Nil5 was grown in 12 ml ofmedium containing 50 M GlnAc to an OD of0.19
before being filtered. The cells were suspended in 12 ml of medium containing 10 M ['4CJGlnAc (specific
activity, 170 juCil,umol). After 0.2 generations (4 min), 4 ml of the culture was filtered, and the cells were
mixed with carrier cells and SLS-inactivated walls prepared as described. The remaining 8 ml was filtered (at
4.5 min), the cells were suspended in 25 ml of unlabeled medium, and growth was allowed to continue.
Samples (10 ml) were filtered at one-half generation (10.5 min) (B) and two generations (41 min) (C) after
resuspension in nonradioactive medium. An equal amount ofcarrier cells (ca. 0.15 g, dry weight) was added
to all samples, and SLS-inactivated walls were prepared. A 6-mg amount of walls from all samples (A, B, and
C) were lysed by the addition of 0.16 ml of a preparation of soluble autolysate (20 mg ofautolyzed wallIml).
For details of lysis and sampling, see Materials and Methods. The insoluble radioactivity (0) and OD 450 (0)
as a percentage of zero time values are plotted against time. One hundred percent radioactivity was 1.25, 2.1,
and 2.2 x 104 dpm/mg of wall, for the samples isolated at zero time (A), at one-half (B), and at two generations
(C), respectively.
Minutes plies that it has bound a higher concentration of
100 10 20 30 40 enzyme, a result expected for a superficial
layer. However, the rate of release of radioac-
tivity relative to overall wall lysis for newly
80 incorporated wall is slower than that of wall
two generations old. This may partly reflect the
physical state of the cell wall preparation. The
inner surface may have bound a lower concen-
60 "x0.D450 tration of added enzyme because of reduced
_ accessibility relative to the outer surface, in
view of the many long, relatively intact tubes
40 present in the cell wall preparation.
1tt4c I believe that the explanation for the differ-
20 I added to a large excess (ca. 0.35 g, dry weight) of
FIG. 7. Lysis of SLS-inactivated cell wall isolated unlabeled carrier cells, and SLS-inactivated walls
from a continuously labeled culture of Nil5 after two were prepared. A 6.0-mg amount of walls was lysed
generations of growth in unlabeled medium. The by the addition of 0.20 ml of a cell wall autolysate (20
growth medium contained 55 pM ['4CJGlnAc (spe- mg of wall/mI) in a total volume of 6.0 ml. Lysis was
cific activity, 5.5 pJAilmol). The culture was filtered measured as described in Materials and Methods.
at an OD of 0.2 after six generations of growth on The OD 450 (0) and insoluble radioactivity (0) as
labeled medium. The cells were suspended at an OD percentage of zero time values are plotted against
of 0.06 and allowed to grow in the chase medium for time; 100% radioactivity was 2.7 x 104 dpmlmg
two generations (40 min) before being refiltered and per ml.
 f. , ~
1146 POOLEY
ent patterns of release of wall of different ages
found in this study lies in the geometry of the
cell wall layer. The present results are consist-
ent with a pattern of flow of cell wall material
during growth (shown schematically in Fig. 8)
in which pulse-labeled mucopeptide, initially
incorporated at or near the membrane, passes
out through the thickness of the cell wall layer,
only reaching the outer surface one and one-
half generations after incorporation, largely in
the form of a layer. The proposed arrangement
(Fig. 8) of cell wall of different ages also pro-
vides an explanation for the failure of pulse-
labeled wall to be turned over in the parent for
one and one-half generations if turnover is re-
stricted to wall which is at the outer surface.
Although it is not possible to be certain about
the precise distribution of the different poly-
mers in the cell wall with the labeling proce-
dure used in the present study, it is clear from
the experiments shown in Fig. 6 that the great
bulk of the pulse label, two generations after
chasing, is released more rapidly than the over-
all-rate of wall lysis. This result, I believe, is
consistent with the view that the major portion
of the mucopeptide synthesized during a pulse
forms part of the outer surface of the cell wall
layer two generations afterwards.
The apparent existence of a similar lag before
turnover of newly incorporated wall in several
species of bacilli (3, 11), and also in Lactobacil-
lus acidophilus (1), may indicate that for these
rod-shaped organisms there is a common pat-
tern of distribution of wall of different ages
within the expanding cell wall layer.
In addition, this arrangement complements
exactly the conclusion that old (labeled) cell
wall forms a superficial layer occupying an ex-
ponentially increasing area during one and
one-half to two generations of growth in unla-
beled medium (13). The geometry of the cell
wall layer proposed here suggests that it is
possible that the fate of most, if not all, wall is
to reach the outer surface, and that by a com-
plementary argument only wall one and one-
half generations old or older forms part of the
outer surface. A higher concentration of autoly-
tic activity bound to old wall (Fig. 7) would not
be expected from a random distribution of old
s
__________ ____________
4-pulse :_i____
________
0 3'
FIG. 8. Schematic model for the position of pulse label within the cell wall layer, at various times (zero,
a
one-half, and two generations) during growth after pulse incorporation.
J. BACTERIOL.
and new wall throughout the cell wall layer
(Fig. 9A), or with new and old wall distributed
in a single, discrete anular band (Fig. 9B), or
even with multiple bands (Fig. 9C) oriented
around either axis of the rod. However, the pref-
erential binding of autolytic activity to old wall
is consistent with a higher surface concentra-
tion of old wall (Fig. 9D). If this interpretation
is correct, it has considerable implications for
the conclusions drawn from the many attempts
to determine the pattern of insertion of new cell
wall material during surface growth in bacilli.
It is implicit in current ideas of how the bacte-
rial cell surface grows that it is possible to
distinguish newly synthesized wall from older
Downloaded from http://jb.asm.org/ on May 18, 2020 by guest
A C
D
_
-._
t
FIG. 9. Four possible modes of distribution of new
and old wall within the cell wall layer. Old (14C-
labeled) wall (a) is shown diagramatically in a sec-
tion through the wall taken along the long axis of the
rod for a continuously labeled culture after two gen-
erations of growth in unlabeled medium. (A) New
and old wall is randomly distributed both along the
long axis and within the wall thickness; (B) new wall
has been inserted in a central localized zone; (C) new
wall has been inserted at many points over the cell
surface, with no variation in concentration of new
and old wall within the thickness of the wall layer;
(D) old wall forms a layer overlying newer wall,
which is the distribution proposed here and in the
accompanying paper (13). The arrows indicate the
position of randomly bound autolytic activity added
during the experiment described in the legend to
Fig. 7.
____________ ---cell wall
.
/-%|~memnbrane
4-cytoplasm
2gen.
VOL. 125, 1976 AGE-DETERMINED LAYERS IN THE CELL WALL
wall by some suitable labeling procedure, by
autoradiography (2, 10), or by means of fluores-
cein-labeled antibody (5, 6, 8). Such procedures
have been used most clearly in streptococcal
species (6) to show a clear distribution of zones
of old and new wall. However, extension of
these methods to the bacilli has failed to yield a
single unambiguous answer (4, 5, 7, 8, 12). The
arrangement of the cell wall proposed in this
work suggests that it is not possible to regard
the entire wall thickness lying between the
membrane and the outer surface as being syn-
thesized within a fairly small fraction of a gen-
eration and which segregates as a discrete unit
during subsequent growth of the surface. The
present results do not allow us to distinguish
between a random or restricted pattern of inser-
tion of new glycan chains, distinct from the
extension of existing chains. However, I believe
that a zonal pattern for new chain insertion has
not been ruled out by the failure to observe
segregation of old, labeled wall using autora-
diography (12) because of the possibility of its
being obscured by the spreading of the old wall.
The process of surface extension discussed
here has to be distinguished from that of septa-
tion in bacilli. It is possible that the septum,
which accounts for about 10% of the total cell
wall, is made up of wall of a very similar age,
and that its formation proceeds in a similar
manner to septum fornation in coccal-shaped
organisms. A different pattern of release of 10%
of the radioactivity would be obscured by the
release of the remaining 90%.
ACKNOWLEDGMENTS
The excellent technical help of Denise Button, and the
many stimulating and critical discussions with H. J.
Rogers, R. F. Rosenberger, and M. G. Sargent are grate-
fully acknowledged.
1147
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