Download Antimicrobial Agents and Chemotherapy

Vol. 2, No. 6
Printed in U.S.A.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1972, p. 485-491
Copyright t 1972 American Society for Microbiology
Inhibition of Peptidoglyean Synthesis by the
Antibiotic Diumycin A
E. J. J. LUGTENBERG,' J. A. HELLINGS, AND G. J. VAN DE BERG
Laboratory for Microbiology, Catharijnesingel 59, Utrecht, The Netherlands
Received for publication 19 October 1972
Diumycin A, a new antibiotic, was found to inhibit cell wall synthesis by Staphylococcus aureus, a phenomenon accompanied by accumulation of uridine-5'-diphosphate-N-acetyl-muramyl-pentapeptide. The antibiotic inhibited in vitro peptidoglycan synthesis by particulate preparations of Bacillus stearothermophilus and
Escherichia coli by preventing the utilization of N-acetyl-glucosamine-N-acetylmuramyl-pentapeptide. In contrast to vancomycin, the antibiotics diumycin,
prasinomycin, moenomycin, 11.837 RP, and enduracidin do not inhibit particulate
D-alanine carboxypeptidase.
The biosynthesis of peptidoglycan, a rigid network consisting of amino sugars and amino acids
(27), is unique to bacteria, and is, therefore, an
ideal target for the action of antibiotics. Phosphonomycin (5) and D-cycloserine (11, 17)
inhibit the biosynthesis of uridine diphosphateN-acetyl-muramyl (UDP-MurNAc)-pentapeptide.
A number of antibiotics cause accumulation of
UDP-MurNAc-pentapeptide in vivo (15, 21).
The development of cell-free systems for peptidoglycan synthesis (1, 2, 22) facilitated studies on
the mechanism of action of these antibiotics.
Penicillin (12, 26, 29), bacitracin (23, 24), vancomycin, ristocetin (2, 7), enduracidin, moenomycin, prasinomycin, and 11.837 RP (15) all
inhibit one of the envelope-bound steps in
peptidoglycan synthesis. Although penicillin and
vancomycin inhibit peptidoglycan synthesis in a
different way, both antibiotics inhibit D-alanine
carboxypeptidase I (8, 9), an enzyme that liberates the ultimate D-alanine residue from UDPMurNAc-pentapeptide in vitro (8).
We recently received a sample of diumycin A,
a new antibiotic isolated from Streptomyces
umbrinus (16). Like the other phosphorus-containing antibiotics prasinomycin (28), moenomycin
(6), and 11.837 RP (D. Mancy et al., Int. Congr.
Microbiol., 9th, Moscow, p. 165, 1966), it is
especially active against gram-positive bacteria,
yeasts, and Mycobacterium bovis, whereas members of the family Enterobacteriaceae are relatively resistant (16). The molecular weight of
diumycin in 90%0 ethanol is 1,600 and in phosphate buffer is 3,200. Similar data have been
I Present address: University of Connecticut Health Center,
Department of Microbiology, Farmington, Conn. 06032.
given for vancomycin (4, 19). Like vancomycin,
it adsorbs ultraviolet light (16). Diumycin A
differs from prasinomycin and moenomycin. It
contains two residues of glucosamine, whereas
each of the latter two antibiotics yield one
equivalent of both glucosamine and 6-deoxyglucosamine under the same hydrolytic conditions
(16).
The present paper describes a study in which
the influences of diumycin A and vancomycin on
peptidoglycan synthesis were compared.
MATERIALS AND METHODS
Bacterial strains. Bacillus stearothermophilus NCTC
10339 and Staphylococcus aureus 524/SC were obtained from P. E. Reynolds, Department of Biochemistry, University of Cambridge, Cambridge,
England. B. cereus strain T and Escherichia coli K-12
strain KMBL-146 were obtained from K. Izaki,
Department of Agriculture, Tokohu University,
Sendai, Japan, and A. Rorsch, Medical Biological
Laboratory, Rijswijk, Z.H., The Netherlands, respec-
tively.
Growth of bacteria. Bacteria were grown with aeration in the complex CGPY medium, containing 0.5%
glucose (12). They were incubated at 37 C, with the
exception of B. stearothermophilus, which was grown
at 55 C. Optical density was measured with a Unicam
SP-600 spectrophotometer at a wavelength of 660
nm. Exponentially growing cells were used for all
experiments.
Buffers. The buffers used had the following compositions (per liter): (A) 5 X 10-2 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride and 10-2
M MgCI2 (pH 7.8); (B) 1.5 M Tris-hydrochloride and
0.3 M MgCI2 (pH 7.8); (C) 0.5 M Tris-hydrochloride,
10-4 M MgCl2, and 10-3 M 2-mercaptoethanol (pH
7.5); and (D) 1 M Tris-hydrochloride and 0.2 M MgC12
(pH 7.5).
485
486
LUGTENBERG, HELLINGS, AND VAN DE BERG
Peptidoglycan synthesis in vivo. Peptidoglycan synthesis in vivo was followed in a modification of the wall
medium CWSM-I (12) containing 10 ,ug of L-alanine
per ml instead of 5 ,ug per ml. The medium was supplemented with glycine (100 ,ug/ml) when S. aureus was
used. If necessary, antibiotics were added at zero time.
For incorporation studies, a sample of the suspension
was added to uniformly labeled 14C-L-alanine (2
,uCi/ml). The unlabeled suspension was incubated
under identical conditions and used to follow the
optical density. Incorporation of label was determined
(i) by counting the acid-precipitable activity or (ii) by
chromatography of heat-inactivated samples as described previously (12, 15). The possibility that radioactive teichoic acid, in addition to peptidoglycan, was
counted as acid-precipitable radioactivity or as a nonmoving component on the chromatogram was discussed previously (15). In a number of experiments,
the suspension was centrifuged to separate the cells
from the medium.
Accumulation of uridine nucleotides. S. aureus cells
were grown and transferred to the cell wall medium
as described for following the peptidoglycan synthesis. The suspension (0.24 mg, dry weight, per ml)
was incubated for 1 hr at 37 C. The cells were harvested, washed, and disrupted by heat treatment.
Macromolecular material was precipitated with trichloroacetic acid. The supernatant fluid was extracted with ether, neutralized, and decreased in
volume under reduced pressure. The amount of Nacetyl-hexosamine was determined by the method of
Reissig et al. (20) as modified by Strominger (25).
Details of this procedure have been described previously (15).
Isolation of particulate enzyme. B. stearothermophphilus particles were isolated as described previously
(15). Small and large envelope fragments were not
separated. The preparation was stored at -20 C in
buffer A. E. coli particles were isolated in the same
way except for the following modifications: (i) the
cells were disintegrated for 10 min; and (ii) the particles that were used to assay D-alanine carboxypeptidase I were suspended in buffer A, whereas
particles that were used in the complete system were
suspended in buffer C.
Cell-free peptidoglycan synthesis. The incubation
mixtures for B. stearothermophilus contained: 5 ,uliters
of UDP - N - acetyl - (glucosamine - 14C), uniformly
labeled (specific activity, 270 IACi/,&mole, 7.7 nmoles/
ml); 5 ,uliters of UDP-MurNAc-pentapeptide (2
,umoles/ml); 5 j.liters of buffer B, 5 ,Aiters of antibiotic
solution or distilled water; and 5 ,liters of particulate
enzyme. The different components were mixed in the
cold and incubated in a water bath at 37 C. Cell-free
peptidoglycan synthesis with E. coli enzyme was performed as described above for B. stearothermophilus
except that buffer D was used instead of buffer B, and
the mixtures were incubated at 30 C. After incubation,
the samples were heat-inactivated for 30 sec at 100 C.
After chromatography and autoradiography (usually
for 1 week), the radioactive spots were excised and
counted (15).
Assay of D-alanine carboxypeptidase I. The incubation mixtures contained: 5 1liters (28,000 counts/min)
ANTIMICROB. AG. CHEMOTHER.
of UDP-MurNAc-L-ala-D-glu-meso-diamino-pimelic
acid-D-ala('4C)-D-ala(14C) (specific activity, 81.8
/hCi//Smole), 5 ,uliters of buffer B, 5 sAliters of antibiotic solution or distilled water, and 10 ,liters of
E. coli particulate enzyme in buffer A, usually containing 10 mg of protein/ml. Incubation was carried
out as described for cell-free peptidoglycan synthesis.
The radioactivity in the alanine spot was taken as a
measure for D-alanine carboxypeptidase activity. In
addition to D-alanine carboxypeptidase I, the preparation also contained an active D-alanine carboxypeptidase 11 (8), as indicated by the nearly complete absence of UDP-MurNAc-tetrapeptide after incubation.
This was shown after isolation of the charcoaladsorbable material from the supernatant fluid of
centrifuged inactivated incubation mixtures. The
UDP was split off by mild hydrolysis in 0.05 N HCI
for 15 min at 100 C (8). MurNAc-tetrapeptide was
separated from MurNAc-pentapeptide in a solvent
system composed of phenol-water (4:1) containing
0.04% 8-hydroxyquinoline (3).
Other methods. Radioactive uridine nucleotide
precursors were isolated by charcoal adsorption (13).
Reference precursors were accumulated (11, 13, 14)
and purified (15) as previously described. Methods for
the separation and identification of radicactive precursors have been published in a previous paper from
this laboratory (13). Protein was determined by the
method of Lowry et al. (10).
Chemicals. Diumycin A (ammonium salt) was a
gift of F. L. Weisenborn, The Squibb Institute for
Medical Research, New Brunswick, N.J. UDP-Nacetyl-glucosamine and vancomycin were obtained
from Boehringer Mannheim NV, Amsterdam, The
Netherlands, and Eli Lilly & Co., Indianapolis, Ind.,
respectively. The origin of the other antibiotics has
been given in a previous paper (15).
Radiochemicals. UDP-N-acetyl - (glucosamine-14C
uniformly labeled) (specific activity, 270 mCi/mmole);
14C-D-alanine, uniformly labeled (specific activity,
40.9 mCi/mmole); and 14C-L-alanine, uniformly
labeled (specific activity, 156 mCi/mmole), were obtained from the Radiochemical Center, Amersham,
England. UDP-MurNAc-L-ala-D-glu-meso-diarnino
pimelic-acid-D-ala(14C)-D-ala("4C) (specific activity,
81.8 mCi/mmole) was prepared by addition of
D-alanyl('4C)-D-alanine(4C) to UDP-MurNAc-tripeptide with the help of crude enzyme of E. coli strain
KMBL-146 as described for the assay of D-alanyl-Dalanine adding enzyme (11), except that excess of
enzyme was used. The product was purified by preparative paper chromatography with the solvents
isobutyric acid-1 M ammonia (5:3, v/v) and ethanol-I
M ammonium acetate, pH 7.2 (5:2, v/v). D-alanyl(14C) -D-alanine("4C) was prepared from uniformly
labeled '4C-D-alanine as described previously (13).
RESULTS
Influence of diumycin on growth. Diumycin at
a concentration of 0.1 ,ug per ml stopped the increase of the optical density of a culture of S.
aureus after some time without causing significant
lysis of the cells (Fig. 1). With increased anti-
VOL. 2, 1972
DIUMYCIN A INHIBITION OF PEPTIDOGLYCAN SYNTHESIS
487
2.0r
B. cereus
1.0
0.8
>1
c
0.L
.2_
6-
0.2
0
30
60
90
time (min)
FIG. 1. Influence of the addition of diumycin A on the growth curves ofexponentially growing cells of S. aureus
and B. cereus. The optical density measured at 660 nm in the absence (0) and in the presence of 0.1 (@) and 1.0
(A) ,ug of diumycin per ml is plotted against time.
biotic concentrations (1 to 100 j,g/ml), the rate of
initial increase in optical density was lower.
Vancomycin, tested at the same concentrations,
gave similar results. The influence of diumycin on
the growth of B. cereus was different. An antibiotic concentration of 0.1 ,ug per ml decreased
the growth rate after 30 min, as a result of lysis
of part of the cells; resumption of growth followed, probably caused by growth of the cells
that escaped the lysis. Gram-stained preparations
showed that these cells were swollen, suggesting
that the envelope was impaired by the presence
of diumycin. Diumymin concentrations between I
and 100 ,ug per ml gave typical lysis curves.
Vancomycin also caused lysis of B. cereus. It is
concluded that the iifluence of diumycin on the
growth of S. aureus and B. cereus is similar to that
of vancomycin, a known cell wall antibiotic.
Cell wail synthesis in vivo. These experiments
were carried out as described previously. The
influence of the presence of diumycin and vancomycin on the incorporation of uniformly labeled
14C-L-alanine from a wall medium into acidprecipitable material of S. aureus was measured.
Only the results for diumycin are given in Fig. 2.
The incorporation was slightly inhibited by
diumycin and vancomycin at concentrations of
0.1 and 1.0 jig per ml, respectively. Tenfold higher
concentrations of both antibiotics decreased the
rate of incorporation dramatically. The optical
density remained constant during the course of
the experiment.
The influence of diumycin and vancomycin on
the synthesis of alanine-containing components
was studied under the same conditions. After
incubation, samples were cooled and centrifuged.
The first supernatant fluid and the resuspended
pellet were heat-inactivated and chromatographed. Autoradiography showed that the supernatant fluid contained the majority of alanine
(RF, 0.65), a weak spot of alanyl-alanine (RF,
0.79), and two weak spots (RF, 0.42 and 0.50),
among which the fast-moving one probably represents pyruvate (11, 13). The pellet contained
macromolecular wall material (RF, 0.0), precursors (RF, 0.2), and a small fraction of alanine.
At RF 0.9, the RF value of lipid intermediates (7),
some radioactivity was present, but it is not certain whethei this activity represents the lipid
intermediates (11). The radioactivities of the main
components after 60 min of incubation are given
in Table 1. Inhibition of cell wall synthesis by each
of the two antibiotics was accompanied by a
dramatic accumulation of uridine nucleotide precursors, to amounts that were roughly 50-fold
higher than that found in the control. Analysis of
the precursors (13) showed that all detectable
radioactivity was present in UDP-MurNAcpentapeptide.
Accumulation of uridine nucleotide precursors
was also shown by assay of the hexosamine content. Samples (100 ml) of S. aureus in a wall
medium without diumycin A and supplemented
with diumycin A in final concentrations of 0.1,
1.0, and 10.0 /Ag/ml contained 0.08, 1.00, 1.12,
and 1.12 ,umoles of uridine nucleotide precursors,
respectively.
Although diumycin caused lysis of growing B.
cereus cells (Fig. 1) and inhibited cell wall syn-
488
LUGTENBERG, HELLINGS, AND
VAN DE
BERG
ANTim[CROB. AG. CHEMOTHER.
which inhibited the incorporation nearly completely. Such a difference of effect on cell wall syn0CD
d(0.1)v(1.0) thesis of S. aureus and B. cereus has been observed for prasinomycin (15), an antibiotic related
to diumycin (16).
The results with S. aureus show that diumycin
inhibits peptidoglycan synthesis, accompanied by
accumulation of UDP-MurNAc-pentapeptide.
This inhibition thus must be due to an action on
one of the membrane-bound steps.
Peptidoglycan synthesis in vitro. Particulate
6
(1.0) v (1o)
of B. stearothermophilus (22) and E.
preparations
(10)
coll (7) contain all enzymes that are required for
the synthesis of cross-linked peptidoglycan. Preparations of the former bacterium imitate the in
1270
0
30
60
90
time(min)
vivo situation better than those of E. coli (15).
FiG. 2. Influence of diumycin and vanccomycin on The effect of various diumycin concentrations on
cell wall synthesis by S. aurues. Exponentrially grow- peptidoglycan synthesis by an extremely active
ing cells were twice washed with wall mediium and re- particulate preparation of B. stearothermophilus
suspended in one volume of the same mecdium. After is shown in Fig. 3. Lipid intermediate and
incubation for 10 min at 37 C, samples of the suspen- peptidoglycan were the only detectable products.
sion were added to prewarmed vials contai)ning a con- Low concentrations of diumycin inhibit peptidomix
1.0-mi
centrated solution of antibiotic.
^
16
0
E
12
8
d
d
0
After
-ing,
samples of the suspensions were added to vial
taining 2.0 pCi of uniformly labeled 14(
The suspensions were incubated at 37 C undrer aeration.
At intervals, the acid-precipitable radioactiviity of O.1-ml
samples was determined (11). The nonlabelec (suspension
was used for optical density measurementsv. The acidprecipitable activities of the samples coi ntaining no
antibiotic (0), diumycin (d), or vancomyIcin (v), in
concentrations indicated in parentheses (imicrograms
per milliliter) are plotted against time.
on-ml
?-L-alani°ne.
thesis by S. aureus in a wall mediunn (Fig. 2
Table 1), it hardly interfered with the iincorporation of L-alanine by B. cereus in the same wall
medium, in contrast to vancomycin (1 ,Ag/ml),
%
I- -
,,--1
glycan synthesis, a phenomenon accompanied by
accumulation of the lipid intermediate. When 1
Ag of diumycin per ml was present, 50% inhibition
of peptidoglycan synthesis was observed. The
radioactivity in the lipid intermediate gradually
decreased with increasing diumycin concentrations. The solubility in
water
of peptidoglycan
synthesized in the absence and presence of
diumycin was the same (14 to 18%). Uncrosslinked peptidoglycan, synthesized by particulate
preparations of E. coli, is soluble in water (7). It
was therefore concluded that diumycin has no
significant influence on the transpeptidation reaction.
TABLE 1. Influence of diumycin and vancomycin on cell wall synthesis by S. aureus"
Counts/min
Addition
Fraction
Cell wall,
RFO.O0
SN
None
P
Diumycin, 1 pug/ml
SN
P
Diumycin,
10jpg/ml
Vancomycin, 10 pg/ml
SN
P
SN
P
85
12,000
72
11,700
44
1,290
56
830
Alanyl-
Lipid inter-
Precursors,
RFO0.2
Alanine,
RFO0.65
alanine,
mediates (?)
86
117
69
238
165
13,300
375
8,800
157,000
2,470
124,000
30,600
149,000
14,300
177,000
8,800
830
80
1,080
150
1,500
260
1,070
210
28
530
21
630
23
830
44
850
RFpO.79
RFpO.9
aExponentially growing cells of S. aureus were centrifuged, washed with, and resuspended in wall
medium. After incubation for 10 min at 37 C, 14C-L-alanine (2 ,Ci/ml) and, if indicated, antibiotic were
added. After 30, 60, and 120 min, samples (0.5 ml) were centrifuged in the cold. The pellet was resuspended in the same medium except that it did not contain labeled alanine. Samples (0.1 ml) of the first
supernatant fluid (SN) and the resuspended pellet (P) were heat-inactivated and chromatographed.
After autoradiography, radioactive compounds were excised and counted. Thjs table gives the results
after incubation for 60 min. Details of the method are described in Materials and Methods.
DIUMYCIN A INHIBITION OF PEPTIDOGLYCAN SYNTHESIS
VOL. 2, 1972
x1o~'
cpml
16i
-I l
a'
a' a'
a'
12
a'
a'
a'
a'
a'
a'
8
a'
489
D-alanine (0.1 mM), the product of carboxypeptidase I, had no influence on the enzyme
activity. Penicillin G and methicillin, added to the
assay in concentrations of 300 j,g/ml, reduced the
liberation of alanine to less than 5 %/. As carboxypeptidase II activity was present, this result shows
that the labeled nucleotide-substrate did not contain a considerable amount of UDP-MurNActetrapeptide. Penicillin G and vancomycin (Fig. 4)
inhibited the liberation of D-alanine by 50%7 in
concentrations of 0.004 and 45 ,ug per ml, respectively, which is in good agreement with the data of
Izaki and Strominger (8) for the soluble enzyme.
Diumycin A, 11.837 RP, moenomycin, prasinomycin, and enduracidin, tested in concentrations
between 10 and 300 j,g per ml, did not inhibit the
carboxypeptidase activity.
DISCUSSION
0
.1
.3
10
30
3
1
Diumycin concentration (lug/mt)
100
FMG. 3. In vitro inhibition of peptidoglycan synthesis
by diumycin A. Particulate enzyme ofB. stearothermophilus was used as described previously in a final protein concentration of 44 ,ug per ml. Incubations were
carried out for I hr at 37 C without and in the presence
of various diumycin concentrations. The radioactivities
of peptidoglycan (solid line) and lipid intermediate
(broken line) are plotted against the final diumycin
concentration.
E. coli preparations are very active in the synthesis of the labeled lipid intermediate, but
peptidoglycan synthesis clearly is the limiting step
in the system. Fifty percent inhibition of peptidoglycan synthesis by particulate preparations of E.
coli K-12 strain KMBL-146 (2 mg of protein per
ml) was obtained at diumycin concentrations of
1 to 3 ,ug per ml. With all diumycin concentrations
tested (0.1 to 30 ,ug/ml), the radioactivity in the
lipid intermediate was higher than in the control.
The differences with the control were less pronounced than with preparations of B.
stearothermophilus, owing to the high activity of
lipid intermediate in the incubation mixture without antibiotic.
D-Alanine carboxypeptidase I. Partly purified
soluble D-alanine carboxypeptidase I of E. coli
strain Y-10 is susceptible to penicillins and vancomycin (8). Because diumycin, as well as 11.837
RP, moenomycin, prasinomycin, and enduracidin
(15), inhibits the same reaction in peptidoglycan
synthesis as vancomycin, we decided to test the
sensitivity of D-alanine carboxypeptidase to these
antibiotics. Particulate preparations of E. coli
strain KMBL-146 were used as described previously. At 30 C, the liberation of alanine was
linear with time for at least 2 hr. The presence of
Like vancomycin, diumycin A inhibits the
growth of S. aureus (Fig. 1) and inhibits cell wall
synthesis by this organism (Fig. 2), events which
are accompanied by accumulation of UDP-
MurNAc-pentapeptide. Although a diumycin
concentration of 0.1 ,g/ml inhibited growth of
S. aureus (Fig. 1), this antibiotic concentration
had no significant influence on the rate of cell wall
synthesis by the same bacterium during incubation
in the wall medium (Fig. 2). This lack of correlation is probably due to the different composition
of the media.
Growing B. cereus cells lysed in the presence of
120
"
100
Z
a 80
X 60
F
Diumycin A
CL
x
0
'U' 40
c
C)
20
-~~
.1 .3 1
3 10 30 100 300
Antibiotic concentration (X9Lg/rn )
0 .0003 .001 .003 .01 .03
FIG. 4. Effect of antibiotics on activity of D-alanine
carboxypeptidase. Enzyme assays were performed as
previously described with a particulate preparation of
E. coli K-12 strain KMBL-146 for 50 miii at 30 C. The
remaining D-alanine carboxypeptidase activity is given
relative to the activity in a sample without antibiotic.
Like diumycin, the antibiotics moenomycin, 11.837
RP, prasinomycin, and enduracidin had no significant
influence on the enzyme activity.
490
LUGTENBERG, HELLINGS, AND VAN DE BERG
1.0 ,ug of diumycin per ml (Fig. 1). In contrast to
vancomycin, but like prasinomycin (15), diumycin
had no detectable influence on cell wall synthesis
by nongrowing B. cereus cells. We have no good
explanation for this phenomenon. One can assume that, in B. cereus, protein synthesis is required for penetration of diumycin and prasinomycin to their target(s).
When the B. cereus cells that escaped lysis in
the presence of 0.1 mg of diumycin per ml (Fig. 1)
were subcultured and grown under identical conditions, the same growth curve was obtained,
showing that this typical curve is not due to two
populations of cells in the original culture. The
same type of growth curve has been found for
E. coli cells grown in the presence of certain concentrations of penicillin or vancomycin (Lugtenberg, unpublished data). We do not have a clearcut explanation of this growth curve. As stated
earlier (Lugtenberg, Ph.D. thesis, State Univ. of
Utrecht, Utrecht, The Netherlands, 1971), one
can speculate that peptidoglycan synthesis occurs
in a particular stage of the cell cycle. Cells that
reach this stage may bind or accumulate relatively
more of the antibiotic than cells that reach this
stage later. The latter cells then escape lysis since
they grew in a relatively lower antibiotic concentration. Another explanation, also based on the
assumption that peptidoglycan biosynthesis occurs in a certain step in the cell cycle, is that after
lysis of part of the bacteria the lysate of these cells
causes partial inactivation of the antibiotic. Inactivation of the well-known antibiotics penicillin
and vancomycin may, for instance, be caused by
penicillinase and UDP-MurNAc-pentapeptide
(18, 19), respectively. However, as conditions or
agents that may inactivate diumycin A are not
known at the moment, we are unable to answer
the question of whether such an explanation
would be reasonable for diumycin A.
Experiments with particulate preparations
showed that diumycin in low concentrations interferes with the ulitization of N-acetyl-glucosamineN-acetyl-muramyl-pentapeptide for peptidoglycan
synthesis. Higher concentrations of the antibiotic
also inhibit the synthesis of the disaccharide-lipid
intermediate (Fig. 3). Diumycin has no influence
on the degree of solubility of peptidoglycan, and
therefore does not interfere with the transpeptidation reaction. Diumycin, prasinomycin, 11.837
RP, enduracidin, and moenomycin do not inhibit
D-alanine carboxypeptidase, in contrast to
vancomycin (Fig. 4). This observation indicates
that the mechanism of action of vancomycin
differs from that of the other five antibiotics. The
inhibition of D-alanine carboxypeptidase by
vancomycin can be explained by the observed
complex formation of the antibiotic with acyl-D-
ANTIMICROB. AG. CHEMOTHER.
ala-D-ala (18). The other five antibiotics probably
do not bind to acyl-D-ala-D-ala. They may inhibit
peptidoglycan synthetase (i) by direct interaction
with the enzyme, (ii) by causing degradation of
the acceptor, although no degradation products
of peptidoglycan could be detected, or (iii) by
distortion of the membrane structure. The latter
explanation is probably true for the ionic detergents sodium dodecyl sulfate, deoxycholate, and
Triton X-100, which inhibit the in vitro utilization
of the disaccharide for peptidoglycan synthesis by
the E. coli particulate system, in contrast to the
nonionic detergent Brij-58, which inhibits peptidoglycan synthesis earlier in the pathway (Lugtenberg, unpublished data).
ACKNOWLEDGMENTS
We thank F. L. Weisenborn for the sample of diumycin A and
Arna van Schijndel-van Damn for expert technical assistance in
part of the experiments.
LITERATURE CITED
1. Anderson, J. S., P. M. Meadow, M. A. Haskin, and J. L.
Strominger. 1966. Biosynthesis of the peptidoglycan of bacterial cell walls. I. Utilization of uridine diphosphate
acetylmuramyl pentapeptide and uridine diphosphate acetyl
glucosamine for peptidoglycan synthesis by particulate
enzymes of Staphylococcus aureus and Micrococcus lysodeikticus. Arch. Biochem. Biophys. 116:487-515.
2. Anderson, J. S., M. Matsuhashi, M. A. Haskin, and J. L.
Strominger. 1967. Biosynthesis of peptidoglycan of bacterial
cell walls. 1I. Phospholipid carriers in the reaction sequence.
J. Biol. Chem. 242:3180-3190.
3. Araki, Y., A. Shimada, and E. Ho. 1966 Effect of penicillin
on cell wall mucopeptide synthesis in an Escherichia coli
particulate system. Biochem. Biophys. Res. Commun. 23:
518-525.
4. Best, G. K., M. K. Grastie, and R. D. McConnell. 1970.
Relative affinity of vancomycin and ristocetin for cell walls
and uridine diphosphate-N-acetylmuramyl pentapeptide. J.
Bacteriol. 102:476-482.
5. Hendlin, D., E. 0. Stapley, M. Jackson, H. Wallick, A. K.
Miller, F. J. Wolf, T. W. Miller, L. Chaiet, F. M. Kahan,
E. L. Foltz, and H. B. Woodruff. 1969. Phosphonomycin,
a new antibiotic produced by strains of Streptomyces.
Science 166:122-123.
6. Huber, G. 1967. Moenomycin. IV. Saurehydrolyse und
Charakterisierung der Spaltprodukte. Liebigs Ann. Chem.
707:170-176.
7. Izaki, K., M. Matsuhashi, and J. L. Strominger. 1968. Biosynthesis of peptidoglycan of bacterial cell walls. XIII.
Peptidoglycan transpeptidase and D-alanine carboxypeptidase, penicillin-sensitive enzymatic reactions in strains of
Escherichia coli. J. Biol. Chem. 243:3180-3192.
8. Izaki, K., and J. L. Strominger. 1968. Biosynthesis of the
peptidoglycan of bacterial cell walls. XIV. Purification and
properties of two D-alanine carboxypeptidases from
Escherichia coli. J. Biol. Chem. 243:3193-3201.
9. Leyh-Bouille, M., J.-M. Ghuysen, M. Nieto, H. R. Perkins,
K. H. Schleifer, and 0. Kandler. 1970. On the Streptomyces
albus G DD carboxypeptidase mechanism of action of
penicillin, vancomycin and ristocetin. Biochemistry 9:
2971-2975.
10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.
Randall. 1951. Protein measurement with the Folin phenol
reagent. J. Biol. Chem. 193:265-275.
11. Lugtenberg, E. J. J. 1972. Studies on Escherichia coli enzymes
VOL. 2, 1972
12.
13.
14.
15.
16.
17.
18.
19.
20.
DIUMYCIN A INHIBITION OF PEPTIDOGLYCAN SYNTHESIS
involved in the synthesis of uridine diphosphate-N-acetylmuramyl-pentapeptide. J. Bacteriol. 110:26-34.
Lugtenberg, E. J. J., and P. G. de Haan. 1971. A simple
method for following the fate of alanine-containing components in murein synthesis of Escherichia coli. Antonie van
Leeuwenhoek J. Microbiol. Serol. 37:537-552.
Lugtenberg, E. J. J., L. de Haas-Menger, and W. H. M.
Ruyters. 1972. Murein synthesis and identification of cell
wall precursors of temperature-sensitive lysis mutants of
Escherichia coli. J. Bacteriol. 109:326-335.
Lugtenberg, E. J. J., and A. van Schijndel-van Dam. 1972.
Temperature-sensitive mutant of Escherichia coli K1 2 with
an impaired D-alanine: D-alanine ligase. J. Bacteriol. 113:
96-104.
Lugtenberg, E. J. J., A. van Schijndel-van Dam, and T. H. M.
van Bellegem. 1971. In vivo and in vitro action of new
antibiotics interfering with the utilization of N-acetylglucosamine-N-acetyl-muramyl-pentapeptide. J. Bacteriol.
108:20-29.
Meyers, E., D. Smith Slusarchyk, J. L. Bouchard, and F. L.
Weisenborn. 1969. The diumycins. New members of an
antibiotic family having prolonged in vivo activity. J.
Antibiot. (Tokyo) 22:490-493.
Neuhaus, F. C. 1968. Selective inhibition of enzymes utilizing
alanine in the biosynthesis of peptidoglycan. Antimicrob.
Ag. Chemother. 1967, p. 304-313.
Nieto, M., and H. R. Perkins. 1971. Modifications of the
acyl-D-alanyl-D-alanine terminus affecting complex-formation with vancomycin. Biochem. J. 123:789-803.
Nieto, M., H. R. Perkins, and P. E. Reynolds. 1972. Reversal
by a specific peptide (diacetyl-arv-L-diaminobutyryl-D-alanylD-alanine) of vancomycin inhibition in intact bacteria and
cell-free preparations. Biochem. J. 126:139-149.
Reissig, J. L., J. L. Strominger, and L. F. Leloir. 1955. A
21.
22.
23.
24.
25.
26.
27.
28.
29.
491
modified colorimetric method for the estimation of Nacetylamino sugars. J. Biol. Chem. 217:959-966.
Reynolds, P. E. 1966. Antibiotics affecting cell-wall synthesis.
Symp. Soc. Gen. Microbiol. 16:47-69.
Reynolds, P. E. 1971. Peptidoglycan synthesis in Bacilli. I.
Effect of temperature on the in vitro system from Bacillus
megateriuns and Bacillus stearothermophiluts. Biochim.
Biophys. Acta 237:239-254.
Siewert, G., and J. L. Strominger. 1967. Bacitracin, an inhibitor of the dephosphorylation of lipidpyrophosphate, an
intermediate in biosynthesis of the peptidoglycan of bacterial cell walls. Proc. Nat. Acad. Sci. U.S.A. 57:767-773.
Stone, K. J., and J. L. Strominger. 1971. Mechanism of action
of bacitracin: complexation of metal ion and C55-isoprenyl
pyrophosphate. Proc. Nat. Acad. Sci. U.S.A. 68:32233227.
Strominger, J. L. 1957. Microbial uiidine-5'-diphosphate-Nacetylamino sugar compounds. I. Biology of the pencillininduced accumulation. J. Biol. Chem. 224:509-523.
Tipper, D. J., and J. L. Strominger. 1968. Biosynthesis of the
peptidoglycan of bacterial cell walls. XII. Inhibition of
cross-linking by penicillins and cephalosporins: studies in
Staphylococcus aureus in vivo. J. Biol. Chem. 243:3169-3179.
Weidel, W., and H. Pelzer. 1964 Bagshaped macromoleculesa new outlook on bacterial cell walls. Advan. Enzymol.
26:193-232.
Weisenborn, F. L., J. L. Bouchard, D. Smith, F. Pancy, G.
Maestrone, G. Miraglia, and E. Meyers. 1967. The prasinomycins: antibiotics containing phosphorus. Nature
(London) 213:1092-1094.
Wise, E. M., and J. T. Park. 1965. Penicillin: its basic site of
action as an inhibitor of a peptide cross-linking reaction in
cell wall mucopeptide synthesis. Proc. Nat. Acad. Sci.
U.S.A. 54:75-81.