FEMS Microbiology Letters 56 (1988) 15-20
Published by Elsevier
15
FEM 03332
Spherical and rod shaped protoplasts
from fl-lactam antibiotic treated cultures of Bacillus subtilis
G. W r i g h t a n d H.J. R o g e r s
The Biological Laboratory, The University, Canterbury, Kent, U.K.
Received 13 June 1988
Accepted 4 July 1988
Key words: Bacillus subtilis; Protoplast; fl-Lactam antibiotic
1. SUMMARY
2. I N T R O D U C T I O N
Addition of fl-lactams to exponentially growing
cultures of an autolytically deficient Bacillus subtills metc3 lyt-2 strain F J6 caused increase in optical density to stop after 1 h when it had about
doubled, and thereafter to remain constant for at
least 6 h. The number of protoplasts to be derived
per unit dry weight of bacteria started to fall when
the antibiotic was added and after I h had reached
50% of the initial value. Also during the first hour
but after a lag of 20-30 min an increasing number
of membrane bound rod shaped protoplasts were
seen among the normal spherical ones. These
swelled to spheres and ultimately burst if the
concentration of sucrose in the suspending fluid
was gradually reduced. These results would be
best explained by rapid inhibition of initiation of
cell division followed by membrane damage to an
increasing proportion of cells.
The primary sites of action of/3-1actam antibiotics are the integral membrane 'penicillin binding
proteins' some or all of which are involved in the
later stages of peptidoglycan synthesis [1]. Inhibition of transpeptidase action is the best defined
result which in itself is likely to halt bacterial
growth. Autolysins hydrolysing peptidoglycan present in almost all bacteria then play a large part in
converting this bacteriostatic effect to a bactericidal one [2,3]. Nevertheless, some bacteria not
possessing detectable autolytic activity (e.g. Group
A Streptococcus pyogenes [4,5] or strains deprived
of autolysis either genetically or phenotypically
are still killed by the antibiotics at an appreciable
rate [6,7] by so-called non-lytic death [8]. Very
soon after the application of the antibiotics to
rapidly growing cultures of such organisms changes
suggesting membrane damage occur [6,9] and even
R N A synthesis may be inhibited and ribonuclease
activated [5]. In the present paper fl-lactam antibiotics are shown to inhibit secondary cell-division
sites in a Bacillus subtilis lyt strain followed shortly
Correspondence to: G. Wright, The BiologicalLaboratory, The
University, Canterbury, Kent CT2 7NJ, U.K.
0378-1097/88/$03.50 © 1988 Federation of European MicrobiologicalSocieties
16
afterwards by such severe membrane damage to a
proportion of the bacterial population that spherical protoplasts can no longer be formed.
ature and were then suitably diluted in the same
fixative solution. All solutions were first passed
through 0.45 ~m membrane filters to remove particles. The results of visual and electronic counting
agreed well.
3. MATERIALS A N D M E T H O D S
3.1. Microorganisms, media, and culture technique
B. subtilis met lyt-2 strain FJ6 [10] and B.
subtilis trpC strain 168 were kept as spore suspensions at - 2 0 ° C . The medium was acid hydrolysed casein, CHSC [11] supplemented by 20
/xg/ml tryptophan for strain 168 or 20 /~g/ml
methionine for strain F J6. Cultures were always
grown in conical or side-arm flasks of five times
their volume at 3 5 ° C in an orbitally shaking
incubator. Inoculation was by placing 1 /~1 of
spore suspension per ml of culture medium into a
flask in the incubator and 8-9 h before the start
of the experiment the required volume of sterile
medium was added aseptically by a time-activated
pump. Exponentially growing cultures were thus
available at the start of the day.
3.2. Protoplast formation
Samples of cultures were mixed with equal
volumes of N a + / K + phosphate buffer (pH 6.6,
0.05 M) containing 1.0 M sucrose, 100 /~g/ml
lysozyme and 2 0 / ~ g / m l DNase and incubated at
35°C for 1 h. Only phase dark spherical protoplasts remained when control cultures were so
treated.
3.3. Protoplast counting
Visual counts were made using phase-contrast
microscopy, with a magnification of 400 and an
'Improved Neubauer' counting chamber (Gallenkamp Ltd.). The number of protoplasts in 10
randomly selected 4 nl volumes was counted, each
volume containing about 100 protoplasts. An electronic Coulter ZF particle counter was also used
with a 30 # m orifice, an aperture current of 32
mA, amplitude of 0.177 and a threshold setting of
16. The protoplasts were first fixed by mixing
suspensions of them with equal volumes of
N a + / K + phosphate buffer (pH 6.6, 0.05 M) containing 0.5 M sucrose and 10% ( v / v ) formalin.
The suspensions stood for 10 min at room temper-
3.4. Measurement of protein
Relative amounts of protein were measured in
protoplasts and in the supernatant fluids from
them by first growing the bacteria in CHSC
medium containing [2(n)-3H]-L-methionine (0.5
/~Ci/ml, 3.8 ~ m o l / m l ) for 4-5 generations. The
protoplast suspensions prepared as described were
centrifuged (12000 × g, 10 min), the supernatant
fluids separated and the protoplasts tysed by addition of p H 6.6 phosphate buffer. Both the supernatant fluids and the lysates were mixed with
equal volumes of 10% ( w / v ) TCA. After 30 rain at
room temperature the precipitates were removed
on 0.45 ~m membrane filters, washed successively
with 0.5% TCA containing 1 m g / m l non-radioactively labelled L-methionine, and 1% ( v / v ) acetic
acid. The membranes and precipitates were dried
at 8 0 ° C overnight and their radioactivity measured by scintillation in toluene containing 0.5%
2,5 diphenyloxazole using a Beckman L.S.7800
scintillation counter.
3.5. Growth
The optical density of cultures at 675 nm wavelength was measured, the results were corrected
for deviations from the Beer-Lambert Law [12],
One unit of optical density was equivalent to 0.5
mg dry weight of bacteria.
3.6. Electron microscopy
Preparations were prefixed with 3% ( w / v )
glutaraldehyde, 3% ( v / v ) formaldehyde, 0.08 M
KC1 and 0.01 M Mg acetate. They were then
post-fixed with 1% ( v / v ) osmium tetroxide, prestained with 0.5% ( w / v ) uranyl acetate and poststained with 0.1 M Pb citrate.
4. RESULTS A N D DISCUSSION
The number of protoplasts derived from exponentially growing cultures of either strain F J6
17
tO0
8o
6O
4O
o
c
2C
~
Time
(h)
Fig. l. Protoplast formation from B. subtilis FJ6 after growth
in C H S C medium containing 0.2 / t g / m l cephalothin. Cultures
were grown exponentially to an OD67 s of 0.15, cephalothin was
added at 0 h and protoplasts were prepared and counted as
described in MATERIALS AND METHODS, from culture samples
taken during the next 4 h of incubation. Both the total protoplast counts ( 0 ) and the n u m b e r of rods ( . ) present are shown
as a percentage of the total protoplast count recorded before
antibiotic addition.
or 168 remained constant in relation to the increasing dry weight of the bacteria. Enumeration
of bacteria themselves in strain F J6 was impossible because B. subtilis lyt strains grow as very long
chains of unseparated cells [10] when the cultures
of this strain were treated with either cephalothin
(0.2/~g/ml) or methicillin (3.0/~g/ml) the number
of protoplasts formed per mg bacterial dry weight
fell by about 50% in the first hour of treatment.
During this time the OD67s doubled but thereafter
remained constant for at least 6 h further incubation [13]. The number of protoplasts that could be
formed continued to fall until after 3 - 4 h it was
reduced to only about 25-30% of the expected
yield per mg of bacterial dry weight (see Fig. 1). It
was at first thought that membrane damage had
precluded protoplast formation and that lysis had
occurred. If so the protein content of the supernatant fluids from protoplast preparations from
antibiotic treated cultures should be higher than
those from control untreated ones. This was not
true. Earlier work [6] had shown that protein was
not lost to the culture fluid by bacteria of F J6
cultures treated with B-lactam antibiotics. The
average protein content per protoplast had how-
ever approximately doubled (1.7 times increase)
during the first hour of treatment of the cultures.
Their size had not changed significantly being as
for protoplasts from control cultures 1.87 _+ 0.2
/~m in diameter. The simplest explanation would
be that cell division was inhibited immediately or
very rapidly by the antibiotics without inhibition
of protein synthesis. Secondary division sites of
Strep. faecalis have been shown to be inhibited by
/3-1actam antibiotics [14]. Examination of transverse sections of the antibiotic treated culture by
electronmicroscopy showed an increasing number
of cells with an incompleted septum between two
completed ones (Fig. 2b). Such cells were effectively twice the volume of normal ones and might
be expected to give rise to protoplasts of twice the
protein content, but also of twice the size. However, the size of protoplasts may within limits be
more a function of internal osmotic pressure than
of amount of material available to them [15].
Among the spherical protoplasts in antibiotic
treated cultures were a number of rod shaped
phase dark forms. These started to appear about
20 min, after adding the/3-1actam to the cultures.
Their proportion of the total was 10-15% after 1 h
and up to 60% after 3 - 4 h (Fig. 1). If buffer not
containing sucrose was gently infused under the
cover-slip of a preparation on a microscope slide,
the rods at first swelled to form spheres, and then
burst. Examination of transverse sections under
the electron microscope (Fig. 2a), confirmed the
rods as membrane bounded bodies very densely
packed with ribosomes. Attempts to separate rod
shaped from spherical protoplasts have not been
successful. Certain chemical proof of the absence
from the former entities of wall material has not
therefore been obtained. When the bacteria were
removed from the antibiotic containing cultures
and grown in CHSC without antibiotic the proportion of rod shaped protoplasts formed was
reduced at the same rate as growth of the bacteria
occurred suggesting that the cells yielding them
were not able to multiply, to repair or to give rise
to spherical protoplasts. It is interesting to note.
that the rod shaped protoplasts started to appear
at the time that membrane damage was first detected [6] and at the time the fluorescent probe
ANS started to interact with bacterial membranes
18
Fig, 2, (a) Transverse section of a rod-shaped protoplast, bounded by membrane only, Bar: 0,25 ~m, (b) Transverse section of
bacteria showing incomplete septum between two completed ones. Bar: 0,25 t~m. In both (a) and (b) the culture had been treated with
0,2 t~g/ml cephatothin for 3 h.
19
[13]. It w o u l d seem p o s s i b l e t h a t the kinetics of
t h e d e v e l o p m e n t of these l a t t e r p h e n o m e n a were
at least p a r t l y a m e a s u r e of p o p u l a t i o n h e t e r o geneity with an i n c r e a s i n g p r o p o r t i o n of cells so
b a d l y m e m b r a n e - d a m a g e d that m u l t i p l i c a t i o n was
n o t possible.
E x p e r i m e n t s w i t h strain 168 were difficult b e cause of the r a p i d lysis t h a t o c c u r r e d after a d d i n g
the a n t i b i o t i c s to e x p o n e n t i a l l y g r o w i n g cultures.
However, d u r i n g the first h o u r of t r e a t m e n t with
a n t i b i o t i c s when little lysis o c c u r r e d r o d s h a p e d
p r o t o p l a s t s f o r m e d a b o u t 3% of the p o p u l a t i o n ,
a n d the n u m b e r of t o t a l p r o t o p l a s t s p e r m g d r y
weight of b a c t e r i a fell to 50% of that in the c o n t r o l
cultures. It w o u l d thus a p p e a r to the b e h a v i o u r of
the fully a u t o l y t i c p a r e n t a l strain was similar to
t h a t of the lyt s t r a i n d u r i n g the early stages of
f l - l a c t a m action b u t cell lysis t h e n s u p e r v e n e d to
greatly accelerate cell death.
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