Vol. 171, No. 3, 1990 September 28, 1990
BIOCHEMICAL
PENICILLIN TAPAS
BINDING
K. SENGUPTA’,
2 Department
Received
August
Eleven
PROTEINS
ANA01
Indian
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1175-1181
OF VIBRIO
N. CHATTERJEEp
CHOLERAE and JYOTIRMOY
DAS’
1 Biophysics Division Institute of Chemical Biology 4 Raja S.C. Mullick Road Calcutta 700032, India
of Food
Technology & Biochemical Jadavpur University Calcutta 700032, India
Engineering
8, 1990
penicillin
binding
proteins
(PBPs)
of
Vibrio
cholerae
have
been
identified
using [ ‘251] labelled p-hydroxybenzyl penicillin (PenX). These proteins are localised in the inner membrane and have molecular weights ranging from 97,000 to 22,000. Neutral hydroxylamine released the labelled PenX from the PBPs and pretreatment with cold benzyl penicillin inhibited labelling completely. The PBP 4 is the most sensitive target for cephaloridine and aztreonam. Cephaloridine also binds to three other high molecular weight PBPs, 1, 2 and 3. Aztreonam, in addition to PBP 4, has affinity for another low molecular weight PBP, PBP 7. Mecillinam has affinity for PBPs 1, 4 and 11. 01990 Academic Press, Inc.
A
large
number
of
membrane-bound
positive
and gram-negative
involved
in the terminal
penicillins, has been major cross
of
total
PBPs
molecular
weight
synthesis
functional
role
1A
1B (Mr
91,000
2 (Mr
66,000)
@,
PBPs enzymes murein
synthesis
endopeptidase
of murein
are
specific and
(4), net
mutants
PBP work
the
in several
penicillin
sensitive
(1). By judicious
defective
(5) and
identified
in the to some and
Mr
controls PBP
3 (Mr
gramenzymes
use of suitable
synthesis
of
PBPs,
of these
proteins
89,000)
constitute
initiation 60,000)
it
(2,3). the
of elongation is involved
in
(6).
PBPs with and
proteins
to assign
cylindrical wall
stages
These
been
and
biosynthetic
of the
bacteria.
have
cephalosporins possible
In Escherichia
PBPs
molecular
present
in
activities
weight
E. coli. (1,7).
less than These
Unlike
PBPs do not appear
*Corresponding
author.
Abbreviations: PBP : Penicillin Sodium dodecyl
binding sulphate
protein, PenX - polyacrylamide
50,000
PBPs the
have
high
to be essential
comprise mainly
molecular for cell
the
major
fraction
DO-carboxypeptidase weight
PBPs,
survival
(8).
penicillin, : p-hydroxybenzyl gel electrophoresis.
the
SDS-PAGE
0006-291X/90 1175
low
:
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
171, No. 3, 1990 In course
cholerae paper
with
BIOCHEMICAL of
our
beta-lactam
documents
studies
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on
the
antibiotics,
the profile
we
of PEPS present
Materials Organisms
and growth
interaction have
of
identified
in this
the
enteropathogen
several
PEPS
v.
and
this
organism.
and Methods
conditions
1. cholerae strain 569B carrying a streptomycin-resistant marker (50 ugl ml) was grown in DIFCO nutrient broth containing 0.18 M NaCl (pH 8.0) (9,lO). 5. coli K-12 was grown in Luria broth (pH 7.0). Cultures were maintained as descxd previously (11). Minimum
inhibitory
concentration
(MC)
MIC of different antibiotics was determined by agar dilution method. Between 300-400 cells were spread on control and antibiotic containing plates (in two fold increments) and MIC was taken to be the least concentration which prevented the appearance of a single colony after incubation for 48 hrs at 37’C. Labelling
of PBPs Labelling
of
PenX
(Gift
from
Dr.
J.T.
Park,
USA) by carrier free Na [ i251] was carried out following Crude cell al (12), using PenX instead of furazlocillin. ‘iTom mid-log phase cultures by differential centrifugation gration of cells (13). Crude cell envelopes (150 ug - 170 ug ded in 50 ul of 50mM phosphate buffer (pH 7.0) were
Tufts
University,
Boston,
the method of Park et envelopes were isolatz after ultrasonic disinteprotein content) suspentreated with 2 ul of
[1251]-PenX and incubated at 30°C (for different time periods) or at 37°C for 10 min. 10 min at 37°C was sufficient to saturate the PBPs of both V. cholerae and g. coli. The labelling was terminated by adding Sarcosyl NL97 71% w/v, final concentration) followed by 20 min of incubation at 25°C. In some cases the incubation mixture was subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) after boiling for 3 min in sample buffer containing 25mM Tris-HCI (pH 6.8), 2% SDS, 5% beta-marcaptoethanol and 0.008% bromophenol blue (14). Alternatively, the Sarcosyl treated membrane preparation was centrifuged at 100,OOOxg for 1 hr (20°C) and the supernatant containing solubilised inner membrane proteins and the pellet containing outer membrane were loaded on the gel separately after boiling in the same buffer. Competition varying ceutical
with
different
beta-&tams
The membrane suspensions were preincubated at 37°C for 10 min with concentrations of Cephaloridine (Sigma), Mecillinam (Gift from Leo PharmaProducts, Denmark) or Aztreonam (Gift from E.R. Squibb and Sons Inc.).
[1251]-PenX was then added and after 10 min of further incubation at 37°C the samples were processed as described above. The final volume of samples loaded on the gel was kept below 70 ul. Detection
of PEPS
Binding of [125 I]-PenX to the membranes was analysed by SDS-PAGE (15). Concentrations of acrylamide and bis-acrylamide were 12% and 0.032% respectively. The gels were stained for 1 hr with 0.1% Coomassie Blue R (Sigma) in 30% methanol, 10% glacial acetic acid and 60% water, destained with the same solution (without the dye), dried under vacuum with heat and exposed to X-ray films (AgfaGaevert, Curix) for 72-96 hrs. Molecular weights of the PBPs were determined by electrophoresing molecular weight marker proteins (Sigma) in the same gel. Densitometric scanning of the autoradiograms were done by a laser densitometer (Ultroscan, L.K.B.). 1176
BIOCHEMICAL
Vol. 171, No. 3, 1990
Release
of bound
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PenX
for 10 min at 37”C, washed Membranes were labelled with [‘25 I]-PenX by centrifugation (lOO,OOOxg, 30 min, 4°C) with 2 ml of 50mM phosphate buffer in 0.1 ml (pli 7.0) containing 1. mg cold benzyl penicillin per ml, resuspended were removed at different of the same buffer and incubated at 37% Aliquots intervals and analysed by SDS-PAGE. Results
and Discussion
PBPs of V. cholefae The [1251]-PenX rials (Fig. An
crude
and
and
cell
analysed
Methods.
1, lane c). The PBP
after
labelling
SDS-PAGE outer
and
proteins
molecular profile of
Thus
v.
were
of these
cell
envelopes of
5696
PBPs ranged isolated with
was
in the from
inner
proteins
PBPs of V. cholerae -
are
with
in Mate-
autoradiogram
97,000
to 22,000.
membrane
(isolation
[‘251]-PenX)
these
labelled
as described
as revealed
when
None
strain
autoradiography
labelled
obtained
crude
all the
cholerae
and
weight
was
autoradiography.
membrane.
of
by SDS-PAGE
Eleven
identical
done
envelope
was
was
analysed
detectable
localised
by
in the
in the CytOplaSmiC
membrane. Preincubation penicillin
or
inhibited with the
ampicillin
the 1 M
label
resolved
of
binding
per
in the
the
cell
ml (both
of [ 1251]-PenX
hydroxylamine
from
crude
(pH
membrane
autoradiogram
with
at 25xMIC) to the
7.0)
for
(16,17). of
envelopes
These
the
cell
30 min
for at
room
(Fig.
ug
10 min
membrane.
observations
SDS-PAGE
50
of
at 37°C
Furthermore, temperature indicated
1, lane
either
that c) are
benzyl
COmpletely
treatment released
all
the proteins indeed
PBPs
of --V. cholerae. a
b
90 6660-
Fig.
1.
PBP profile of V. cholerae and E. coli. [ ‘251]-PenX, Cell envelopes %f mrae $dT -- coli were labelled with inner membrane was isolated and analysed by SDS-PAGE as described in of c. 7coli K-12. Lane b : Materials and Methods. Lane a : PBP profile indicate the drfferent PBPs in Densitometric scan of lane c. Numbers 5698. Numbers on order or decreasing Mr. Lane c : PSPs of V. -- cholerae sides
of
lanes
a and
c represent
molecular
1177
weight
in kilodalton.
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
scan
autoradiogram
A densitometric the
low
molecular
cholerae. 40%
The
of
the
weight
Mr
40,000
protein
total
label.
Under
PBPs
in v. cholerae,
(Fig.
1, lane
resolved coli
observed
for --E. coli
PBPs
gel.
The
in the
release
the
kinetics
by terminating
towards
weight
PBP
7 even
halflife
tial
release
labelling,
10 min
of
senting
PBPs
PenX
from cell
bands
1. nam
in 5.
reported
envelopes
at 30°C
was
incubation
and analysing
affinity
was
of
of
different
for
example,
3 and
PBPs for
have
the
PBPs
was
of v.
analysed
no
loss of
reduction (Fig. PenX,
d). Hence,
there
was
a differen-
after
incubated
only
to
3), similar labelled
completion
of
in presence
of
in the
to that
suggesting
molecular high
intensity
cell
weight molecular
of bands reported
membranes
that
the
or
and autoradio-
low
intensity in the
subse-
stability
by SDS-PAGE
restricted
that
the
cholerae and
less than
of
reflects
whether
washed
samples
having
been high
cephaloridine The
and found cephaloridine.
reported
is known
at a very
(3,19).
determined
ug/ml
from
beta-lactams
mecillinam
PBPs even
weight were
with
beta-lactams
PBP
2, lane
Labelling
repre-
for other
with
process
Sarcosyl of release
(1).
preincubation For
significantly 10 min.
PBPs
almost
bound
was
rate.
for
of [125 I]-PenX
release
at a slower Fig.
were
[ 1251]-PenX
there
in nature
10
times
Treatment
the
cholerae and
PBPs
to
c and
different
envelopes
11 was observed
to other
molecular
not
v. cholerae
The
To examine
7, 9 and
Several
specific
11
of
out at 37°C PenX
3), a significant
was enzymatic
of E. coli.
cell
during
labelled
1, lane
(Fig.
inhibited
of
times
at 30°C
complex. the
of bound
(17,18).
to bind
were
PBP profile
PBPs
to crude
also
carried
of bound
PBP-PenX
3). While
PBP
is
coli
weight
the
of
(Fig.
(Fig.
were
labelled
Release
Effect
with
about
was analysed
6 of 5.
molecular
autoradiography.
incubation
and at different
(Fig.
and
11 were
at 37°C
of release
penicillin
NL97
low
for
identification
envelope
5 and
of different
at different
of
experiments
of the
weight
affinity
different
30 min
the
organisms
was
after
after
the
labelling
7, 9 and
Rate
PBPs
of
of [‘25 I]-PenX
by SDS-PAGE
PBPs
labelling
graphy.
PBPs
in v.
accounts
the cell
that
present
2). Two high molecular weight PBPs, 1 125 I]-PenX and the binding was not relatively low affinity for [ after 15 min of incubation (Fig. 2, lanes c and d). The low mole-
even
cular
cold
allowed
b) showed
PBPs
b) alone
g. coli
is in agreement
relative
binding
PenX
4, exhibited
complete
the
of
envelopes
cell
observed
the
of [‘251J-PenX
examined
quent
16 and
1, lane
of
1, lane
when
abundance
study
(Fig.
bulk
which
resolved
1A and
present
the and
7, Fig.
conditions
relative
To determine
PBPs
(P6P
the
(17).
Binding_and PenX,
constitute
6 PBPs were
a). The
in the
of the
PBPs
RESEARCH COMMUNICATIONS
affinity have
concentration in
values
of
these
to be 5 ug/ml
for
The
these
1178
effect
of
for
(19).
general
for
high
to be specific
has
MIC
high to
known
affinity PBP
Similarly,
high
2 and for
beta-lactams beta-lactams
PBPs fails
aztreonam
affinity
mecillinam
PBPs
for
high
towards and
aztreoon
the
Vol.
BIOCHEMICAL
171, No. 3, 1990
a :
b
c
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
d e c
03
02 Fig.
2.
Kinetics of binding of PenX to PBPs of V. cholerae. Cell envelopes of v. cholerae 5696 wGe%%&ted with [ ‘251]-PenX at 30°C for 2 (a), 5 (b), 15 (c) and 30 min (d). Inner membranes were then isolated and analysed by SDS-PAGE as described in Materials and Methods. PEPS with relatively low affinity for PenX are indicated by numbers and arrows.
Fig.
3.
Release of PenX from Pt3Ps of V. cholerae. Release of labelled PenX with timefrom the inner membrane of V. cholerae 5696 was examined after 0 (a), 15 (b) and 30 min (c) of incubation of cell envelope in presence of cold benzyl penicillin (1 mg/ml) at 37°C. Numbers indicate PBPs which show progressive release of label.
Be
Fig.
4.
b
c
d
e
Effect of preincubation with mecillinam (A), aztreonam and cephaloridine (B) on binding of PenX to PBPs of V. cholerae. A. Preincubation with 0 (a), 5 Tb)m) and 50 ug (d) mecillinam per ml at 37%. Preincubation with 0 (a), 5 (b) and 50 ug (c) of aztreonam per ml 0. at 37°C. Lanes d (10 us/ml) and e (50 ug/ml) show the effect of different concentrations of cephaloridine. Numbers indicate PBPs which were most affected.
1179
Vol.
171, No. 3, 1990
cellular
BIOCHEMICAL
morphology
reported
for
spheroplast
of
E.
v.
coli
(8).
formation
mecillinam
the
binding
molecular
was
antibiotic
was
highly
E.
PBP
(compare
c&,
where
effect
with to
on
even
binding
cholerae
at
to three
PBPs.
relatively
Aztreonam a
(18).
PBPs,
In i.
4 and 7 (Fig. (at
the
this
PBP
is apparently
dance
MIC
(17).
coli
been
PBPs
1, 2, 3 and
target
for
inhibited
this even
common
same
time
penicillins
the
same MIC
as
level,
bodies
by
binding per
PBP
4 and
PBP
Fig.
4A).
of to
affected
only
the
The
inhibition
was
raised
the
PBP
inhibitory
2 (17),
binding
in v.
of PenX
PBP
3 of
binding
of
PenX
to
g. two
of PBP 7 by aztreo-
in view
of
5 and 6 in size
and
E.
to to
for
the
of
1 was of
in contrast
the
of labelling
PBPs
PBP
specific
surprising
PBPs
11.
mecillinam
highly
Inhibition
to
10 ug/ml
Thus,
inhibited
be
was
PenX
about
mecillinam
weight
of
concentration
a,
affec-
1 and 4, and the low
ml,
restricted
to
markedly
coli
the
fact
that
relative
are
abun-
known
to
be
PBPs
of
for PBP 3 (6,19).
having
affinity
binding 46,
of lanes
the
for
PenX
high
to v.
d and e). The
PenX-PBP
molecular
weight
cholerae
high
PBP
4 was the
4 complex
molecular
formation
weight
most
sensitive
was completely
at 1xMIC.
The many
is
to E. coli
and
the
shaped
PBPs,
mecillinam
cholerae
molecular
antibiotic
at
mecillinam
concentrations
b and c).
specific
4 (Fig.
the
antibiotic
in v.
the
be
coccal
with
lane
reported
this
low
to
culture
weight
of
penicillin
similar
affected
high
has
by antibiotics
(8),
d with
lanes
phase
mecillinam
4 when
concentration
Cephaloridine, 5.
While
binding
very
level)
These
unaffected
4A).
low
46,
nam
found
cephaloridine,
molecular
PBP
cholerae,
log
high 5 ug
labelled
and
suspension
c and
with
of
by
inhibit
for lanes
to
membrane
11 (Fig.
required
checked
by aztreonam.
to two
pronounced
50 ug/ml
induced
of
by preincubation
was added
forms
of PenX
weight
inhibited was
When
and filamentous Preincubation
ted
cholerae
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
present
study
features significant
shows
with
that
those
differences
although
of
other
in
the
PBPs
of v.
microorganisms,
their
response
to
cholerae
there different
are
share at
the
PBP-specific
and cephalosporins. Ac know1 edgments
This No.
investigation
was
BT/03/TF/26/009/88),
senior
research
Govt.
of India.
fellowship
supported Govt. from
by of
the
India. Council
Department One of
of
of
us (T.K.S.)
Scientific
and
Biotechnology is
(Grant
a recipient
Industrial
Research,
References
1. 2.
Waxman, D.J. and Strominger, J.L. (1983) Broom-Smith, J.K. (1985) J. Gen. Microbial. 1180
Ann. Rev. Biochem. 131. 2115-2118.
52, 825-869.
of
Vol. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
171, No. :3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Spratt, B.G. (1980) Philos. Trans. R. Sot. London Ser. B 289, 273-283. Suzuki, H., Van Heijenoort, Y., Tamura, T., Mizoguchi, J., Hirota, Y. and Van Heijenoort, J. (1980) FEBS Lett. 110, 245-249. Sprat& B.G., Jabanputra, V., Zimmerman% W. (1977) Antimicrob. Agents Chemother. 12. 406-409. Botta, G.A. and Park, J.T. (1981) J. Bacterial. 145, 333-340. M., Ghuysen, J.M., Coyette, J., Linder, R., Pollock, J.J., Nguyen-Disteche, Salton, M.R.J., Kim, K.S., Perkins, H.R. and Reynolds, P.E. (1974) Eur. J. Biochem. 41, 439-446. Spratt, B.G. (1975) Proc. Natl. Acad. Sci. USA 72, 2999-3003. Lohia, A., Chatterjee, A.N. and Das, J. (1984) J. Gen. Microbial. 130, 2027-2033. Paul, S., Sen, A.K., Banerjee, N., Chatterjee, A.N. and Das, J. (1990) Biochem. Biophys. Res. Commun. 169, 116-122. Roy, N.K., Das, G., Balganesh, T.S., Dey, S.N., Ghosh, R.K. and Das, J. (1982) *J. Gen. Microbial. 126, 1927-1932. Park, J.T. and Mahadevan, S. (1988) J. Bacterial. 170, 3750-3751. Lohia, A., Majumder, S., Chatterjee, A.N. and Das, J. (1985) J. 6acter iol. 163, 1158-l 166. Spratt, B.G. and Pardee, A.B. (1975) Nature (London) 254, 516-517. Laemmli, U.K. (1970) Nature (London) 227, 680-685. Blumberg, P.M. and Strominger, J.L. (1972) Proc. Natl. Acad. Sci. USA 69, 3751-3755. Spratt, B.G. (1977) Eur. J. Biochem. 72, 341-352. Georgopapadakou, N.H. (1988) in Antimicrob. Agents Ann. Vol. 3 (Ed. P.K. Peterson and V. Verhoef), Elsevier. Schmidt, L.S., Botta, G. and Park, J.T. (1981) J. Bacterial. 145, 632-637.
1181
BIOCHEMICAL
PENICILLIN TAPAS
BINDING
K. SENGUPTA’,
2 Department
Received
August
Eleven
PROTEINS
ANA01
Indian
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1175-1181
OF VIBRIO
N. CHATTERJEEp
CHOLERAE and JYOTIRMOY
DAS’
1 Biophysics Division Institute of Chemical Biology 4 Raja S.C. Mullick Road Calcutta 700032, India
of Food
Technology & Biochemical Jadavpur University Calcutta 700032, India
Engineering
8, 1990
penicillin
binding
proteins
(PBPs)
of
Vibrio
cholerae
have
been
identified
using [ ‘251] labelled p-hydroxybenzyl penicillin (PenX). These proteins are localised in the inner membrane and have molecular weights ranging from 97,000 to 22,000. Neutral hydroxylamine released the labelled PenX from the PBPs and pretreatment with cold benzyl penicillin inhibited labelling completely. The PBP 4 is the most sensitive target for cephaloridine and aztreonam. Cephaloridine also binds to three other high molecular weight PBPs, 1, 2 and 3. Aztreonam, in addition to PBP 4, has affinity for another low molecular weight PBP, PBP 7. Mecillinam has affinity for PBPs 1, 4 and 11. 01990 Academic Press, Inc.
A
large
number
of
membrane-bound
positive
and gram-negative
involved
in the terminal
penicillins, has been major cross
of
total
PBPs
molecular
weight
synthesis
functional
role
1A
1B (Mr
91,000
2 (Mr
66,000)
@,
PBPs enzymes murein
synthesis
endopeptidase
of murein
are
specific and
(4), net
mutants
PBP work
the
in several
penicillin
sensitive
(1). By judicious
defective
(5) and
identified
in the to some and
Mr
controls PBP
3 (Mr
gramenzymes
use of suitable
synthesis
of
PBPs,
of these
proteins
89,000)
constitute
initiation 60,000)
it
(2,3). the
of elongation is involved
in
(6).
PBPs with and
proteins
to assign
cylindrical wall
stages
These
been
and
biosynthetic
of the
bacteria.
have
cephalosporins possible
In Escherichia
PBPs
molecular
present
in
activities
weight
E. coli. (1,7).
less than These
Unlike
PBPs do not appear
*Corresponding
author.
Abbreviations: PBP : Penicillin Sodium dodecyl
binding sulphate
protein, PenX - polyacrylamide
50,000
PBPs the
have
high
to be essential
comprise mainly
molecular for cell
the
major
fraction
DO-carboxypeptidase weight
PBPs,
survival
(8).
penicillin, : p-hydroxybenzyl gel electrophoresis.
the
SDS-PAGE
0006-291X/90 1175
low
:
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
171, No. 3, 1990 In course
cholerae paper
with
BIOCHEMICAL of
our
beta-lactam
documents
studies
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on
the
antibiotics,
the profile
we
of PEPS present
Materials Organisms
and growth
interaction have
of
identified
in this
the
enteropathogen
several
PEPS
v.
and
this
organism.
and Methods
conditions
1. cholerae strain 569B carrying a streptomycin-resistant marker (50 ugl ml) was grown in DIFCO nutrient broth containing 0.18 M NaCl (pH 8.0) (9,lO). 5. coli K-12 was grown in Luria broth (pH 7.0). Cultures were maintained as descxd previously (11). Minimum
inhibitory
concentration
(MC)
MIC of different antibiotics was determined by agar dilution method. Between 300-400 cells were spread on control and antibiotic containing plates (in two fold increments) and MIC was taken to be the least concentration which prevented the appearance of a single colony after incubation for 48 hrs at 37’C. Labelling
of PBPs Labelling
of
PenX
(Gift
from
Dr.
J.T.
Park,
USA) by carrier free Na [ i251] was carried out following Crude cell al (12), using PenX instead of furazlocillin. ‘iTom mid-log phase cultures by differential centrifugation gration of cells (13). Crude cell envelopes (150 ug - 170 ug ded in 50 ul of 50mM phosphate buffer (pH 7.0) were
Tufts
University,
Boston,
the method of Park et envelopes were isolatz after ultrasonic disinteprotein content) suspentreated with 2 ul of
[1251]-PenX and incubated at 30°C (for different time periods) or at 37°C for 10 min. 10 min at 37°C was sufficient to saturate the PBPs of both V. cholerae and g. coli. The labelling was terminated by adding Sarcosyl NL97 71% w/v, final concentration) followed by 20 min of incubation at 25°C. In some cases the incubation mixture was subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) after boiling for 3 min in sample buffer containing 25mM Tris-HCI (pH 6.8), 2% SDS, 5% beta-marcaptoethanol and 0.008% bromophenol blue (14). Alternatively, the Sarcosyl treated membrane preparation was centrifuged at 100,OOOxg for 1 hr (20°C) and the supernatant containing solubilised inner membrane proteins and the pellet containing outer membrane were loaded on the gel separately after boiling in the same buffer. Competition varying ceutical
with
different
beta-&tams
The membrane suspensions were preincubated at 37°C for 10 min with concentrations of Cephaloridine (Sigma), Mecillinam (Gift from Leo PharmaProducts, Denmark) or Aztreonam (Gift from E.R. Squibb and Sons Inc.).
[1251]-PenX was then added and after 10 min of further incubation at 37°C the samples were processed as described above. The final volume of samples loaded on the gel was kept below 70 ul. Detection
of PEPS
Binding of [125 I]-PenX to the membranes was analysed by SDS-PAGE (15). Concentrations of acrylamide and bis-acrylamide were 12% and 0.032% respectively. The gels were stained for 1 hr with 0.1% Coomassie Blue R (Sigma) in 30% methanol, 10% glacial acetic acid and 60% water, destained with the same solution (without the dye), dried under vacuum with heat and exposed to X-ray films (AgfaGaevert, Curix) for 72-96 hrs. Molecular weights of the PBPs were determined by electrophoresing molecular weight marker proteins (Sigma) in the same gel. Densitometric scanning of the autoradiograms were done by a laser densitometer (Ultroscan, L.K.B.). 1176
BIOCHEMICAL
Vol. 171, No. 3, 1990
Release
of bound
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
PenX
for 10 min at 37”C, washed Membranes were labelled with [‘25 I]-PenX by centrifugation (lOO,OOOxg, 30 min, 4°C) with 2 ml of 50mM phosphate buffer in 0.1 ml (pli 7.0) containing 1. mg cold benzyl penicillin per ml, resuspended were removed at different of the same buffer and incubated at 37% Aliquots intervals and analysed by SDS-PAGE. Results
and Discussion
PBPs of V. cholefae The [1251]-PenX rials (Fig. An
crude
and
and
cell
analysed
Methods.
1, lane c). The PBP
after
labelling
SDS-PAGE outer
and
proteins
molecular profile of
Thus
v.
were
of these
cell
envelopes of
5696
PBPs ranged isolated with
was
in the from
inner
proteins
PBPs of V. cholerae -
are
with
in Mate-
autoradiogram
97,000
to 22,000.
membrane
(isolation
[‘251]-PenX)
these
labelled
as described
as revealed
when
None
strain
autoradiography
labelled
obtained
crude
all the
cholerae
and
weight
was
autoradiography.
membrane.
of
by SDS-PAGE
Eleven
identical
done
envelope
was
was
analysed
detectable
localised
by
in the
in the CytOplaSmiC
membrane. Preincubation penicillin
or
inhibited with the
ampicillin
the 1 M
label
resolved
of
binding
per
in the
the
cell
ml (both
of [ 1251]-PenX
hydroxylamine
from
crude
(pH
membrane
autoradiogram
with
at 25xMIC) to the
7.0)
for
(16,17). of
envelopes
These
the
cell
30 min
for at
room
(Fig.
ug
10 min
membrane.
observations
SDS-PAGE
50
of
at 37°C
Furthermore, temperature indicated
1, lane
either
that c) are
benzyl
COmpletely
treatment released
all
the proteins indeed
PBPs
of --V. cholerae. a
b
90 6660-
Fig.
1.
PBP profile of V. cholerae and E. coli. [ ‘251]-PenX, Cell envelopes %f mrae $dT -- coli were labelled with inner membrane was isolated and analysed by SDS-PAGE as described in of c. 7coli K-12. Lane b : Materials and Methods. Lane a : PBP profile indicate the drfferent PBPs in Densitometric scan of lane c. Numbers 5698. Numbers on order or decreasing Mr. Lane c : PSPs of V. -- cholerae sides
of
lanes
a and
c represent
molecular
1177
weight
in kilodalton.
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
scan
autoradiogram
A densitometric the
low
molecular
cholerae. 40%
The
of
the
weight
Mr
40,000
protein
total
label.
Under
PBPs
in v. cholerae,
(Fig.
1, lane
resolved coli
observed
for --E. coli
PBPs
gel.
The
in the
release
the
kinetics
by terminating
towards
weight
PBP
7 even
halflife
tial
release
labelling,
10 min
of
senting
PBPs
PenX
from cell
bands
1. nam
in 5.
reported
envelopes
at 30°C
was
incubation
and analysing
affinity
was
of
of
different
for
example,
3 and
PBPs for
have
the
PBPs
was
of v.
analysed
no
loss of
reduction (Fig. PenX,
d). Hence,
there
was
a differen-
after
incubated
only
to
3), similar labelled
completion
of
in presence
of
in the
to that
suggesting
molecular high
intensity
cell
weight molecular
of bands reported
membranes
that
the
or
and autoradio-
low
intensity in the
subse-
stability
by SDS-PAGE
restricted
that
the
cholerae and
less than
of
reflects
whether
washed
samples
having
been high
cephaloridine The
and found cephaloridine.
reported
is known
at a very
(3,19).
determined
ug/ml
from
beta-lactams
mecillinam
PBPs even
weight were
with
beta-lactams
PBP
2, lane
Labelling
repre-
for other
with
process
Sarcosyl of release
(1).
preincubation For
significantly 10 min.
PBPs
almost
bound
was
rate.
for
of [125 I]-PenX
release
at a slower Fig.
were
[ 1251]-PenX
there
in nature
10
times
Treatment
the
cholerae and
PBPs
to
c and
different
envelopes
11 was observed
to other
molecular
not
v. cholerae
The
To examine
7, 9 and
Several
specific
11
of
out at 37°C PenX
3), a significant
was enzymatic
of E. coli.
cell
during
labelled
1, lane
(Fig.
inhibited
of
times
at 30°C
complex. the
of bound
(17,18).
to bind
were
PBP profile
PBPs
to crude
also
carried
of bound
PBP-PenX
3). While
PBP
is
coli
weight
the
of
(Fig.
(Fig.
were
labelled
Release
Effect
with
about
was analysed
6 of 5.
molecular
autoradiography.
incubation
and at different
(Fig.
and
11 were
at 37°C
of release
penicillin
NL97
low
for
identification
envelope
5 and
of different
at different
of
experiments
of the
weight
affinity
different
30 min
the
organisms
was
after
after
the
labelling
7, 9 and
Rate
PBPs
of
of [‘25 I]-PenX
by SDS-PAGE
PBPs
labelling
graphy.
PBPs
in v.
accounts
the cell
that
present
2). Two high molecular weight PBPs, 1 125 I]-PenX and the binding was not relatively low affinity for [ after 15 min of incubation (Fig. 2, lanes c and d). The low mole-
even
cular
cold
allowed
b) showed
PBPs
b) alone
g. coli
is in agreement
relative
binding
PenX
4, exhibited
complete
the
of
envelopes
cell
observed
the
of [‘251J-PenX
examined
quent
16 and
1, lane
of
1, lane
when
abundance
study
(Fig.
bulk
which
resolved
1A and
present
the and
7, Fig.
conditions
relative
To determine
PBPs
(P6P
the
(17).
Binding_and PenX,
constitute
6 PBPs were
a). The
in the
of the
PBPs
RESEARCH COMMUNICATIONS
affinity have
concentration in
values
of
these
to be 5 ug/ml
for
The
these
1178
effect
of
for
(19).
general
for
high
to be specific
has
MIC
high to
known
affinity PBP
Similarly,
high
2 and for
beta-lactams beta-lactams
PBPs fails
aztreonam
affinity
mecillinam
PBPs
for
high
towards and
aztreoon
the
Vol.
BIOCHEMICAL
171, No. 3, 1990
a :
b
c
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
d e c
03
02 Fig.
2.
Kinetics of binding of PenX to PBPs of V. cholerae. Cell envelopes of v. cholerae 5696 wGe%%&ted with [ ‘251]-PenX at 30°C for 2 (a), 5 (b), 15 (c) and 30 min (d). Inner membranes were then isolated and analysed by SDS-PAGE as described in Materials and Methods. PEPS with relatively low affinity for PenX are indicated by numbers and arrows.
Fig.
3.
Release of PenX from Pt3Ps of V. cholerae. Release of labelled PenX with timefrom the inner membrane of V. cholerae 5696 was examined after 0 (a), 15 (b) and 30 min (c) of incubation of cell envelope in presence of cold benzyl penicillin (1 mg/ml) at 37°C. Numbers indicate PBPs which show progressive release of label.
Be
Fig.
4.
b
c
d
e
Effect of preincubation with mecillinam (A), aztreonam and cephaloridine (B) on binding of PenX to PBPs of V. cholerae. A. Preincubation with 0 (a), 5 Tb)m) and 50 ug (d) mecillinam per ml at 37%. Preincubation with 0 (a), 5 (b) and 50 ug (c) of aztreonam per ml 0. at 37°C. Lanes d (10 us/ml) and e (50 ug/ml) show the effect of different concentrations of cephaloridine. Numbers indicate PBPs which were most affected.
1179
Vol.
171, No. 3, 1990
cellular
BIOCHEMICAL
morphology
reported
for
spheroplast
of
E.
v.
coli
(8).
formation
mecillinam
the
binding
molecular
was
antibiotic
was
highly
E.
PBP
(compare
c&,
where
effect
with to
on
even
binding
cholerae
at
to three
PBPs.
relatively
Aztreonam a
(18).
PBPs,
In i.
4 and 7 (Fig. (at
the
this
PBP
is apparently
dance
MIC
(17).
coli
been
PBPs
1, 2, 3 and
target
for
inhibited
this even
common
same
time
penicillins
the
same MIC
as
level,
bodies
by
binding per
PBP
4 and
PBP
Fig.
4A).
of to
affected
only
the
The
inhibition
was
raised
the
PBP
inhibitory
2 (17),
binding
in v.
of PenX
PBP
3 of
binding
of
PenX
to
g. two
of PBP 7 by aztreo-
in view
of
5 and 6 in size
and
E.
to to
for
the
of
1 was of
in contrast
the
of labelling
PBPs
PBP
specific
surprising
PBPs
11.
mecillinam
highly
Inhibition
to
10 ug/ml
Thus,
inhibited
be
was
PenX
about
mecillinam
weight
of
concentration
a,
affec-
1 and 4, and the low
ml,
restricted
to
markedly
coli
the
fact
that
relative
are
abun-
known
to
be
PBPs
of
for PBP 3 (6,19).
having
affinity
binding 46,
of lanes
the
for
PenX
high
to v.
d and e). The
PenX-PBP
molecular
weight
cholerae
high
PBP
4 was the
4 complex
molecular
formation
weight
most
sensitive
was completely
at 1xMIC.
The many
is
to E. coli
and
the
shaped
PBPs,
mecillinam
cholerae
molecular
antibiotic
at
mecillinam
concentrations
b and c).
specific
4 (Fig.
the
antibiotic
in v.
the
be
coccal
with
lane
reported
this
low
to
culture
weight
of
penicillin
similar
affected
high
has
by antibiotics
(8),
d with
lanes
phase
mecillinam
4 when
concentration
Cephaloridine, 5.
While
binding
very
level)
These
unaffected
4A).
low
46,
nam
found
cephaloridine,
molecular
PBP
cholerae,
log
high 5 ug
labelled
and
suspension
c and
with
of
by
inhibit
for lanes
to
membrane
11 (Fig.
required
checked
by aztreonam.
to two
pronounced
50 ug/ml
induced
of
by preincubation
was added
forms
of PenX
weight
inhibited was
When
and filamentous Preincubation
ted
cholerae
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
present
study
features significant
shows
with
that
those
differences
although
of
other
in
the
PBPs
of v.
microorganisms,
their
response
to
cholerae
there different
are
share at
the
PBP-specific
and cephalosporins. Ac know1 edgments
This No.
investigation
was
BT/03/TF/26/009/88),
senior
research
Govt.
of India.
fellowship
supported Govt. from
by of
the
India. Council
Department One of
of
of
us (T.K.S.)
Scientific
and
Biotechnology is
(Grant
a recipient
Industrial
Research,
References
1. 2.
Waxman, D.J. and Strominger, J.L. (1983) Broom-Smith, J.K. (1985) J. Gen. Microbial. 1180
Ann. Rev. Biochem. 131. 2115-2118.
52, 825-869.
of
Vol. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
171, No. :3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Spratt, B.G. (1980) Philos. Trans. R. Sot. London Ser. B 289, 273-283. Suzuki, H., Van Heijenoort, Y., Tamura, T., Mizoguchi, J., Hirota, Y. and Van Heijenoort, J. (1980) FEBS Lett. 110, 245-249. Sprat& B.G., Jabanputra, V., Zimmerman% W. (1977) Antimicrob. Agents Chemother. 12. 406-409. Botta, G.A. and Park, J.T. (1981) J. Bacterial. 145, 333-340. M., Ghuysen, J.M., Coyette, J., Linder, R., Pollock, J.J., Nguyen-Disteche, Salton, M.R.J., Kim, K.S., Perkins, H.R. and Reynolds, P.E. (1974) Eur. J. Biochem. 41, 439-446. Spratt, B.G. (1975) Proc. Natl. Acad. Sci. USA 72, 2999-3003. Lohia, A., Chatterjee, A.N. and Das, J. (1984) J. Gen. Microbial. 130, 2027-2033. Paul, S., Sen, A.K., Banerjee, N., Chatterjee, A.N. and Das, J. (1990) Biochem. Biophys. Res. Commun. 169, 116-122. Roy, N.K., Das, G., Balganesh, T.S., Dey, S.N., Ghosh, R.K. and Das, J. (1982) *J. Gen. Microbial. 126, 1927-1932. Park, J.T. and Mahadevan, S. (1988) J. Bacterial. 170, 3750-3751. Lohia, A., Majumder, S., Chatterjee, A.N. and Das, J. (1985) J. 6acter iol. 163, 1158-l 166. Spratt, B.G. and Pardee, A.B. (1975) Nature (London) 254, 516-517. Laemmli, U.K. (1970) Nature (London) 227, 680-685. Blumberg, P.M. and Strominger, J.L. (1972) Proc. Natl. Acad. Sci. USA 69, 3751-3755. Spratt, B.G. (1977) Eur. J. Biochem. 72, 341-352. Georgopapadakou, N.H. (1988) in Antimicrob. Agents Ann. Vol. 3 (Ed. P.K. Peterson and V. Verhoef), Elsevier. Schmidt, L.S., Botta, G. and Park, J.T. (1981) J. Bacterial. 145, 632-637.
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