Microbiol
. Immunol.
Vol. 21 (11), 631-638, 1977
Thermolabile Repression of Cephalosporinase Synthesis in Citrobacter freundii Tetsuo
SAWAI, Sachio NAKAJIMA, Toshiro and Saburo YAMAGISHI
MOROHOSHI,
Faculty of PharmaceuticalSciences,Chiba University,Chiba (Received for publication, July 13, 1977)
Abstract An unusual regulatory system of cephalosporinase synthesis in Citrobacterfreundii has been found. When the bacteria are grown at 20 C, the cephalosporinase is synthesized as a typical inducible enzyme and benzylpenicillin acts as an effective inducer. The enzyme, however, is synthesized in the absence of the inducer at growth temperatures above 25 C. When the growth temperature is shifted from 20 C to 37 C, the induction of enzyme synthesis is observed after about one half of the organism doubling time, but it does not occur in the presence of chloramphenicol. The reverse control mutants, the enzyme constitutive synthesis of which is markedly depressed by benzylpenicillin, were isolated from the C. freundii wild strain. The possibility that the enzyme synthesis is governed by a regulatory system analogous to the its mutant of the lac operon in Escherichiacoli was suggested.
Beta-lactamase (EC 3.5.2.6) has been found to be produced by a wide range of bacterial species and the production of the enzyme is one of the main mechanisms of bacterial resistance to beta-lactam antibiotics. Beta-lactamase can be classified into two groups, penicillinase and cephalosporinase, according to the characteristics of their substrate specificity. In a previous study (11), we observed that the cephalosporinase type is the most common among beta-lactamases of gram-negative rod bacteria. These cephalosporinases are, in general, inducible and beta-lactam antibiotics act as inducers. Citrobacterfreundii has the ability to produce a cephalosporinase as a speciesspecific beta-lactamase (11). Enzymological properties of the cephalosporinase were reported previously (12). In the course of our study on the cephalosporinases, we found that the cephalosporinase of a C. freundii strain was synthesized as a constitutive enzyme when the bacteria were grown at 37 C, but the enzyme behaved as an inducible enzyme when the bacteria were grown at 20 C. This paper describes the unique properties of the cephalosporinase synthesis in C.freundii. This study was presented in part at the 2nd Symposium on Antibiotic Resistance, Bratislava, Czechoslovakia, 5th to 8th June 1974 and at International Symposium on Bacterial Resistance, Tokyo, Japan, 24th to 26th October 1974. 631
632
T. SAWAI
MATERIALS
ET AL
AND METHODS
Bacterial strains. C. freundii GN346 is a clinical isolate and a high producer of cephalosporinase, and shows high resistance to all the beta-lactam antibiotics tested (11, 12). The strain GN346/16 is a mutant strain which lost the ability to produce high cephalosporinase activity, and was selected after treatment of parental strain GN346 with N-methyl-N'-nitro-N-nitrosoguanidine (NG) (12). Beta-lactam antibiotics and media. Cephalosporins and penicillins were kindly provided by the following pharmaceutical companies : Cephaloridine and cephalexin by the Torii Pharmaceutical Co., Tokyo, Japan; cephalothin by the Shionogi Pharmaceutical Co., Osaka, Japan; cefazolin by the Fujisawa Pharmaceutical Co., Osaka, Japan ; cephalosporin C and cephaloglycin by Glaxo Laboratories, England; and benzylpenicillin, ampicillin and 6-aminopenicillanic acid by the Meiji Seika Co., Tokyo, Japan. All of the media used were products of Eiken Chemical Co., Tokyo, Japan. Determination of cephalosporinase. Cephalosporinase activity was assayed iodometrically by a modification of Perret's method (10). The enzyme reaction was carried out in 0.1 Mphosphate buffer (pH 6.8) containing 8 mm cephaloridine as substrate at 30 C. The hydrolyzed substrate was calculated on the basis of one mole being equivalent to two moles (4 atoms) of iodine (1). One unit of the enzyme activity was defined as the activity which hydrolyzes one ,umole of the substrate in min under the conditions described above. RESULTS
Effect
of
Growth
Temperature
Cephalosporinase perature. various
1 shows with
or
without
is that
of
a
synthesis mode
apparently
50,
was
The
by
the
substrate cephalothin,
enzyme
than
activity addition
crude
presence
or
profiles
for
those the the
400
six
of
,ƒÊg/ml
20
C.
of
the
cells
the
usual
by
25
and
the
increasing
for 20
tem-
grown
of
at
cephalotempera-
inducible
temperature 30,
growth
organisms mode
With the
37,
was
The
20
1 hr
grown C
type the
to
an
C. freundii
C were
cephalosporins
maximum
about
50,
extracted
from were
and cefazolin,
two
higher
found
the
concentration
cephalosporinase at
was
after
benylpenicillin
cephaloglycin,
the
induced
cells at
inducer
preparations
absence
cephalexin,
at
in of
enzyme
at
the
C,
enzyme. from growth
times
of
growth less
optimal
20
GN346
affected
of
At
varied
freundii
respectively.
concentration
markedly
C. was
activities inducer.
doubling
in
strain
inducible
The
The
bacterial
the
synthesis
min,
Synthesis
C. freundii
typical
enzyme
C.
200
the
cephalosporinase
type.
37 to
further
Four in
to
160
to
the
units
the
inducer
C was
ever,
30
and
inhibitory 20
of
the
constitutive
70
in
Table
the
strain
Cephalosporinase
temperatures
sporinase ture,
on
synthesis
the
temperatures. to
first
not
activity
increase
at How-
to
about
20
C or
respect
to
12
addition. cells
compared penicillins, cephalosporin
grown
at
with i.e.,
cephaloridine,
C,
benzylpenicillin
37 their
C
CEPHALOSPORINASE
Table 1.
SYNTHESIS
IN C. FREUNDII
633
Cephalosporinase activity in C. freundii GN346 grown at various temperatures
A bacterial culture growing exponentially in nutrient broth at an indicated temperature was diluted 5-fold with a fresh medium to give an optical density of about 0.1 at 610 nm. In the case of cultivation in the presence of an inducer, the indicated amount of benzylpenicillin was added to the culture. The diluted culture was incubated with shaking at the indicated temperature until its optical density at 610 nm reached about 0.5, and the growth of the bacteria was stopped by chilling the culture in an ice-bath. After the culture optical density was measured to estimate bacterial dry weight per ml, the chilled culture was treated for 2 min with an ultrasonic disintegrator and assayed for its cephalosporinase activity. The concentration of the inducer ration which allowed uninhibited each growth temperature.
was the maximum concentgrowth of the bacteria at
and ampicillin. No essential differences in the substrate profiles were observed among the four cephalosporinase preparations. Inductionof Cephalosporinase Synthesisby Penicillins Benzylpenicillin and 6-aminopenicillanic acid are known as effective inducers for cephalosporinases of gram-negative bacteria (2, 4, 5, 6). Efficiency of the penicillins as the inducer on the cephalosporinase synthesis in C. freundii GN346 was examined. As a result, benzylpenicillin was found to be more effective with respect to inducibility than 6-aminopenicillanic acid (Fig. 1). Figure 2 shows the kinetics of induced cephalosporina sesynthesis in the organism growing at 20 C. A linear increase in the enzyme activity was observed 45 min after addition of the inducer. Inductionof Cephalosporinase Synthesisby Increasing the Temperature The results given in Table 1 suggested the possibility that cephalosporinase synthesis is governed by a regulatory system which is very thermolabile. Such an assumption was supported by the experimental results shown in Fig. 3. When the
634
T. SAWAI
Fig.
1.
Effect
of
ET AL
benzylpenicillin
and
6-aminopenicil-
lanic acid on cephalosporinase synthesis in C. .freundii GN346 growing at 20 C. A bacterial culture growing exponentially in a nutrient broth at 20 C was diluted with prewarmed (20 C) nutrient broth to give an optical density of about 0.1 at 610 nm. The diluted culture was incubated with shaking at the same temperature in the presence or absence of penicillin for 3 hr, and the specific cephalosporinase activity of the cells was assayed as described in Table 1. The curves show results with different initial concentrations of penicillin. acid.
organism 20
in
C to
the
37
the C,
increase
added the
start
the of
during
to
large
tried
i-
the
C were
isolated.
the
mutant
strain
the
shifted
chemical
no 37
a growth was
inducer.
When of
increase
in
500
enzyme
i.e.,
the
60
of
min
after
chloramphenicol
was
simultaneously
with
,ƒÊg/ml
the
temperature
detected
activity
was
observed
C.
20
iC in
obtained
higher
mutants,
the
absence
from
these
cephalosporinase of
from
activity
Synthesis
at
Some
was
concentration
so-called
enzyme was
slightly
20
at
Cephalosporinase
mutant
showed
any final
increase,
isolate
of
typical
which
phase
●, 6-aminopenicillanic
cephalosporinase
of a
incubation
to
amounts
the
give
temperature
Affecting
We
in
addition
culture
the
growth
increase
subsequent
Mutations
no
exponential an
without
to
○, benzylpenicillin;
them
GN346/101
showed
listed
in
an Table
early
attempts, than
is
a
which
inducers.
activity unexpected 2
mutants
of chemical
but the
and
produce However,
mutant
strains
parental
curious
representative
strain
property, of
such
at and
mutant
strains. Isolation GN346 20 (pH
C,
in were 6.0).
of the
the
harvested The
strain
exponential
washed
GN346/101 growth
by
centrifugation cells
were
phase,
was
carried
out
as
which
were
grown
in
washed
once
and mutagenized
with
300
follows. heart
with ,ƒÊg/ml
The infusion
0.1 of
M citrate NG
in
cells
of
broth
at
buffer the
buffer
CEPHALOSPORINASE
Fig.
2.
Induction
zylpenicillin
20
C
was
the
reached
optical
the
assayed
30
C for
for
cells 48
water)
hr
were was
at
and
(pH 7.0) Colonies
30
20
This
spread C.
treated
with
purified one
mutant
iodine/potassium white haloes examined. 6 •~
cells
103
at
the
covered
The surviving
of
a
was
portions
of times
cephalosporithe
culture in
was
Table
1.
survivors.
The
0.5% with in
(8 mm/60 mm) cephalosporinase
frequency
nm
to
indicated
benzylpenicillin
iodide indicative
610
culture
as described
10%
and
incubation
and of
A
growth
containing
were
1%
at
C. broth
broth
inducer,
about agar
colonies of
the
density
yield
a solution
per
to and
activity
the
infusion
resulting
and
of
optical
disrupting
heart
added
enzyme
procedure
on
The
containing developing
picked, about
min.
were
The
after
was
removed
20
temperature.
culture
mid-exponential
were of
activity.
the
the
addition
culture
measurement
nase
nized
of
(in
fresh same
of 400 ƒÊg/ml
After
induced
the
635
by ben-
at
a nutrient
with
at
density 0.5
concentration
continued.
in
10-fold
benzylpenicillin
final
growing
overnight
shaking
about
phase),
at
diluted
synthesis
GN346
grown
with
When
for
C. freundii
culture
incubated
IN C. FREUNDII
of cephalosporinase
in
bacterial at
SYNTHESIS
of
starch soft-agar
0.067 at
appearance
NG-mutageand
incubated
(0.5%
M phosphate
agar
room temperature. production at 20 of
the
in
buffer
C
mutant
cells.
The strain GN346/101 behaved as a microconstitutive mutant at 20 C, and increased its enzyme activity with an increase in the growth temperature. When the mutant strain was grown at 30 C, benzylpenicillin, which is the most effective inducer for the enzyme induction in the wild strain, stopped the enzyme synthesis in the mutant strain (Fig.4A). The partially purified enzymes prepared from the cells of GN346/101 grown at 20 C and 30 C were examined concerning their substrate profiles, Km values to ampicillin and cephaloridine, and heat stabilities. No differences were found in these properties between the mutant and parent strains. Mutant strain GN346/16-110 is a constitutive mutant derived from strain GN346/16 and was isolated as follows. The cells of GN346/16 were treated with NG
636
T. SAWAI
Fig.
3.
Induction
of
ET AL
cephalosporinase
synthesis
by
in-
creasing the growth temperature in C.freundii GN346. A bacterial culture grown overnight in nutrient broth at 20 C was diluted 10-fold with prewarmed (20 C) broth, and incubated with shaking at 20 C. When the optical density of the culture at 610 nm reached about 0.4 (in the mid-exponential growth phase), the growth temperature was increased from 20 C to 37 C. Portions of the culture were removed at the indicated times and assayed concerning optical density and specific cephalosporinase activity as described in Table 1. Table 2.
Cephalosporinase activity of mutant strains derived from C. freundii GN346 grown at 20 C or 37 C in the
The
organism
was grown
presence
or absence
in a nutrient
broth
of benzylpenicillin
at the indicated
temperature
in
the presence or absence of the inducer, and assayed for its specific cephalosporinase activity as described in Table 1. The concentrations of the inducer did not have obvious inhibitory effects on the bacterial growth which was followed by measurements of turbidity.
in
a similar
at
20
C for
manner
to
5 hr.
The
containing
200
incubation
at
about
one
,ƒÊg/ml 20
mutant
that
described
above,
mutagenized of
cephaloridine.
C for
two
per
107
days cells
and
cells
were plated.
were The
picked,
cultured spread colonies purified
in fresh on
heart
appearing and
examined.
heart
infusion
infusion on
the
broth
agar plates We
plates after
obtained
CEPHALOSPORINASE
SYNTHESIS
IN
C. FREUNDH
637
A
Fig.
4.
The
relationship
between
B
specific
cephalosporinase
activity
and
the
inducer
(benzylpenicillin) concentration shown by the reverse control type mutants of C. freundii GN 346 grown at various temperatures. The experimental procedures and conditions for the bacterial growth, the induction of cephalosporinase and assay of the enzyme activity are the same as those described in Table 1. The concentrations of benzylpenicillin used did not show obvious inhibitory effects on the normal growth of the organisms. The curves show the results with different initial concentrations of the inducer at a growth temperature of 20c(●
〉, and
30
c(○)。
A,
c. freundii
GN346/101;
B,C.freecndii
GN346/16-110.
The mutant GN346/16-110, as shown in Table 2, is able to produce high cephalosporinase activity without any chemical inducer over a growth temperature range of 20 to 37 C. In Fig. 4B, the enzyme activities of the mutant strain growing in the presence of various concentrations of benzylpenicillin are indicated. Benzylpenicillin acted like a strong corepressor for the synthesis of the enzyme in the mutant at both 20 C and 30 C. No differences in heat stability and other enzymological properties could be detected between the enzyme from GN346/16-110 and the wild enzyme. DISCUSSION
It is known that many cephalosporinases produced by gram-negative rod bacteria are inducible enzymes, and beta-lactam antibiotics act as the inducer. However, little is known about the induction mechanism. Garber and Friedman suggested on the basis of induction kinetics by penicillin that the induction mechanism of cephalosporinase in the pseudomonads is similar to the beta-galactosidase system of Escherichiacoli (4). If the regulatory system for cephalosporinase in C.freundii is analogus to that for lac operon enzymes, the most probable explanation for this unique property of cephalosporinase synthesis may be that the enzyme synthesis is regulated by a highly thermolabile repressor similar to the repressor protein of the its mutant of the lac operon (7,8). The mutant strains GN346/101 and GN346/16-110 are so-called reverse control mutants. The characteristics of the mutants are also similar to those reported for reverse control mutants, the i gene mutants, of the lac operon
638
T. SAWAI
ET AL
(9). Recently, De Graaff et al isolated a C. freundii Hfr donor strain and F-prime factor (3). This has enabled genetical analysis in the species. C. freundii GN346 and its mutants will offer advantages for understanding the molecular mechanism of cephalosporinase induction in gram-negative bacteria. C. freundii GN346 is a clinical isolate and not a specifically isolated mutant for particular experimental purposes in vitro. The thermolabile property of the cephalosporinase synthesis seems to be common in the C. freundii species because other strains of the species, which were randomly selected from our stock strains highly resistant to ampicillin and cephaloridine, possesed this unique property. In our experiments, we could not find a similar regulatory system among the cephalosporinase-producing strains belonging to other species of gram-negative enteric bacteria. We have as yet no information concerning the reason for such a unique regulatory system from the teleological point of view. When C. freundii GN346 was measured for single-cell resistance to cephaloridine and to ampicillin on heart infusion agar plates containing a two-fold dilution of the antibiotics at 37 C, we could not observe any differences in the resistance levels between cells precultured at 20 C and 37 C, respectively. Therefore, the thermolabile regulatory system does not appear to play an important role in bacterial resistance to antibiotics. REFERENCES 1)
Alicino, and
2)
J.F.
Ayliffe,
G.A.
Gen. 3)
4)
De
Graaff,
Hfr
males
Horiuchi, Mol.
108:
Horiuchi,
3:
703-704. and
G.L.,
and
coli.
10 )
Perret,
C. J.
11 )
Sawai,
T.,
activity
A.H.
van
J.
Inducible
1974.
Leeuwenhoek
of
acid
Gram-negative
Isolation
40:
and
Gen.
the
bacteria.
J.
penicillinase
64: in
and
characterization
of
161-170.
resistance
Microbiol.
Inducible ƒÀ-lactamase S.,
and
Novick,
A.
J.
Sadler,
J.R.
Mol.
in
Novick,
1965.
A.
Studies
58:
S.,
and
1954. Iodometric
of Pseudomonas
aeruginosa
to
343-352.
Proteus
morganii.
Biochem.
Biophys.
Res.
Enterobacter.
1961.
A
J.
Gen.
Microbiol.
temperature-sensitive
of a thermolabile
49:
277-285.
regulatory
repressor.
Biochim.
system.
Biophys.
J.
Acta
T.,
Microbiol.
cephalosporanic
acid Proc.
in
12:
Mitsuhashi,
6th
Mutational
inversion
of
control
of
the
lactose
operon
of
1-28. assay
of ƒÀ-lactamases
J.
1971.
Biol.
Mitsuhashi,
Comparison
bacteria.
penicillinase
1970. ƒÀ-Lactamase
1963.
1967.
T.,
T.,
Myers,
Sawai,
6-aminopenicillanic
687-696.
Escherichia
Japan.
penicillins,
43-48.
T.D.
Horiuchi,
synthetic
Stouthamer,
cephalosporins.
J.M.T.
Biol.
and Antonie
J.
and
13:
and
648-649. and
P.C., freundii.
Friedman,
Hamilton-Miller,
Hennessey,
12 )
Kreuning,
and
of natural 33:
119-126.
Citrobacter
penicillins
7)
Chem.
Cephalosporinase
40:
N.,
assay
Anal.
1965.
J.,
Garber,
6)
9)
J.
in
Commun.
8)
C.
Microbiol.
various 5)
1961. Iodometric
cephalosporin
of
penicillinase.
Yamagishi,
S.
gram-negative
Nature 1968. rod
(London)
Drug
174:1012-1013.
resistance
bacteria
resistant
of
enteric
bacteria.
XIV.
to ƒ¿-aminobenzylpenicillin.
423-434. S.,
and Internat.
and
Yamagishi,
S.
6-aminopenicillanic Congr.
Chemotherapy
1970.
Stability
acid
to I:
of
various
cephalosporinases
derivatives from
of
7-amino-
gram-negative
410-415.
Requests for reprints should be addressed to Dr. Tetsuo Sawai, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Chiba 280, Japan.
. Immunol.
Vol. 21 (11), 631-638, 1977
Thermolabile Repression of Cephalosporinase Synthesis in Citrobacter freundii Tetsuo
SAWAI, Sachio NAKAJIMA, Toshiro and Saburo YAMAGISHI
MOROHOSHI,
Faculty of PharmaceuticalSciences,Chiba University,Chiba (Received for publication, July 13, 1977)
Abstract An unusual regulatory system of cephalosporinase synthesis in Citrobacterfreundii has been found. When the bacteria are grown at 20 C, the cephalosporinase is synthesized as a typical inducible enzyme and benzylpenicillin acts as an effective inducer. The enzyme, however, is synthesized in the absence of the inducer at growth temperatures above 25 C. When the growth temperature is shifted from 20 C to 37 C, the induction of enzyme synthesis is observed after about one half of the organism doubling time, but it does not occur in the presence of chloramphenicol. The reverse control mutants, the enzyme constitutive synthesis of which is markedly depressed by benzylpenicillin, were isolated from the C. freundii wild strain. The possibility that the enzyme synthesis is governed by a regulatory system analogous to the its mutant of the lac operon in Escherichiacoli was suggested.
Beta-lactamase (EC 3.5.2.6) has been found to be produced by a wide range of bacterial species and the production of the enzyme is one of the main mechanisms of bacterial resistance to beta-lactam antibiotics. Beta-lactamase can be classified into two groups, penicillinase and cephalosporinase, according to the characteristics of their substrate specificity. In a previous study (11), we observed that the cephalosporinase type is the most common among beta-lactamases of gram-negative rod bacteria. These cephalosporinases are, in general, inducible and beta-lactam antibiotics act as inducers. Citrobacterfreundii has the ability to produce a cephalosporinase as a speciesspecific beta-lactamase (11). Enzymological properties of the cephalosporinase were reported previously (12). In the course of our study on the cephalosporinases, we found that the cephalosporinase of a C. freundii strain was synthesized as a constitutive enzyme when the bacteria were grown at 37 C, but the enzyme behaved as an inducible enzyme when the bacteria were grown at 20 C. This paper describes the unique properties of the cephalosporinase synthesis in C.freundii. This study was presented in part at the 2nd Symposium on Antibiotic Resistance, Bratislava, Czechoslovakia, 5th to 8th June 1974 and at International Symposium on Bacterial Resistance, Tokyo, Japan, 24th to 26th October 1974. 631
632
T. SAWAI
MATERIALS
ET AL
AND METHODS
Bacterial strains. C. freundii GN346 is a clinical isolate and a high producer of cephalosporinase, and shows high resistance to all the beta-lactam antibiotics tested (11, 12). The strain GN346/16 is a mutant strain which lost the ability to produce high cephalosporinase activity, and was selected after treatment of parental strain GN346 with N-methyl-N'-nitro-N-nitrosoguanidine (NG) (12). Beta-lactam antibiotics and media. Cephalosporins and penicillins were kindly provided by the following pharmaceutical companies : Cephaloridine and cephalexin by the Torii Pharmaceutical Co., Tokyo, Japan; cephalothin by the Shionogi Pharmaceutical Co., Osaka, Japan; cefazolin by the Fujisawa Pharmaceutical Co., Osaka, Japan ; cephalosporin C and cephaloglycin by Glaxo Laboratories, England; and benzylpenicillin, ampicillin and 6-aminopenicillanic acid by the Meiji Seika Co., Tokyo, Japan. All of the media used were products of Eiken Chemical Co., Tokyo, Japan. Determination of cephalosporinase. Cephalosporinase activity was assayed iodometrically by a modification of Perret's method (10). The enzyme reaction was carried out in 0.1 Mphosphate buffer (pH 6.8) containing 8 mm cephaloridine as substrate at 30 C. The hydrolyzed substrate was calculated on the basis of one mole being equivalent to two moles (4 atoms) of iodine (1). One unit of the enzyme activity was defined as the activity which hydrolyzes one ,umole of the substrate in min under the conditions described above. RESULTS
Effect
of
Growth
Temperature
Cephalosporinase perature. various
1 shows with
or
without
is that
of
a
synthesis mode
apparently
50,
was
The
by
the
substrate cephalothin,
enzyme
than
activity addition
crude
presence
or
profiles
for
those the the
400
six
of
,ƒÊg/ml
20
C.
of
the
cells
the
usual
by
25
and
the
increasing
for 20
tem-
grown
of
at
cephalotempera-
inducible
temperature 30,
growth
organisms mode
With the
37,
was
The
20
1 hr
grown C
type the
to
an
C. freundii
C were
cephalosporins
maximum
about
50,
extracted
from were
and cefazolin,
two
higher
found
the
concentration
cephalosporinase at
was
after
benylpenicillin
cephaloglycin,
the
induced
cells at
inducer
preparations
absence
cephalexin,
at
in of
enzyme
at
the
C,
enzyme. from growth
times
of
growth less
optimal
20
GN346
affected
of
At
varied
freundii
respectively.
concentration
markedly
C. was
activities inducer.
doubling
in
strain
inducible
The
The
bacterial
the
synthesis
min,
Synthesis
C. freundii
typical
enzyme
C.
200
the
cephalosporinase
type.
37 to
further
Four in
to
160
to
the
units
the
inducer
C was
ever,
30
and
inhibitory 20
of
the
constitutive
70
in
Table
the
strain
Cephalosporinase
temperatures
sporinase ture,
on
synthesis
the
temperatures. to
first
not
activity
increase
at How-
to
about
20
C or
respect
to
12
addition. cells
compared penicillins, cephalosporin
grown
at
with i.e.,
cephaloridine,
C,
benzylpenicillin
37 their
C
CEPHALOSPORINASE
Table 1.
SYNTHESIS
IN C. FREUNDII
633
Cephalosporinase activity in C. freundii GN346 grown at various temperatures
A bacterial culture growing exponentially in nutrient broth at an indicated temperature was diluted 5-fold with a fresh medium to give an optical density of about 0.1 at 610 nm. In the case of cultivation in the presence of an inducer, the indicated amount of benzylpenicillin was added to the culture. The diluted culture was incubated with shaking at the indicated temperature until its optical density at 610 nm reached about 0.5, and the growth of the bacteria was stopped by chilling the culture in an ice-bath. After the culture optical density was measured to estimate bacterial dry weight per ml, the chilled culture was treated for 2 min with an ultrasonic disintegrator and assayed for its cephalosporinase activity. The concentration of the inducer ration which allowed uninhibited each growth temperature.
was the maximum concentgrowth of the bacteria at
and ampicillin. No essential differences in the substrate profiles were observed among the four cephalosporinase preparations. Inductionof Cephalosporinase Synthesisby Penicillins Benzylpenicillin and 6-aminopenicillanic acid are known as effective inducers for cephalosporinases of gram-negative bacteria (2, 4, 5, 6). Efficiency of the penicillins as the inducer on the cephalosporinase synthesis in C. freundii GN346 was examined. As a result, benzylpenicillin was found to be more effective with respect to inducibility than 6-aminopenicillanic acid (Fig. 1). Figure 2 shows the kinetics of induced cephalosporina sesynthesis in the organism growing at 20 C. A linear increase in the enzyme activity was observed 45 min after addition of the inducer. Inductionof Cephalosporinase Synthesisby Increasing the Temperature The results given in Table 1 suggested the possibility that cephalosporinase synthesis is governed by a regulatory system which is very thermolabile. Such an assumption was supported by the experimental results shown in Fig. 3. When the
634
T. SAWAI
Fig.
1.
Effect
of
ET AL
benzylpenicillin
and
6-aminopenicil-
lanic acid on cephalosporinase synthesis in C. .freundii GN346 growing at 20 C. A bacterial culture growing exponentially in a nutrient broth at 20 C was diluted with prewarmed (20 C) nutrient broth to give an optical density of about 0.1 at 610 nm. The diluted culture was incubated with shaking at the same temperature in the presence or absence of penicillin for 3 hr, and the specific cephalosporinase activity of the cells was assayed as described in Table 1. The curves show results with different initial concentrations of penicillin. acid.
organism 20
in
C to
the
37
the C,
increase
added the
start
the of
during
to
large
tried
i-
the
C were
isolated.
the
mutant
strain
the
shifted
chemical
no 37
a growth was
inducer.
When of
increase
in
500
enzyme
i.e.,
the
60
of
min
after
chloramphenicol
was
simultaneously
with
,ƒÊg/ml
the
temperature
detected
activity
was
observed
C.
20
iC in
obtained
higher
mutants,
the
absence
from
these
cephalosporinase of
from
activity
Synthesis
at
Some
was
concentration
so-called
enzyme was
slightly
20
at
Cephalosporinase
mutant
showed
any final
increase,
isolate
of
typical
which
phase
●, 6-aminopenicillanic
cephalosporinase
of a
incubation
to
amounts
the
give
temperature
Affecting
We
in
addition
culture
the
growth
increase
subsequent
Mutations
no
exponential an
without
to
○, benzylpenicillin;
them
GN346/101
showed
listed
in
an Table
early
attempts, than
is
a
which
inducers.
activity unexpected 2
mutants
of chemical
but the
and
produce However,
mutant
strains
parental
curious
representative
strain
property, of
such
at and
mutant
strains. Isolation GN346 20 (pH
C,
in were 6.0).
of the
the
harvested The
strain
exponential
washed
GN346/101 growth
by
centrifugation cells
were
phase,
was
carried
out
as
which
were
grown
in
washed
once
and mutagenized
with
300
follows. heart
with ,ƒÊg/ml
The infusion
0.1 of
M citrate NG
in
cells
of
broth
at
buffer the
buffer
CEPHALOSPORINASE
Fig.
2.
Induction
zylpenicillin
20
C
was
the
reached
optical
the
assayed
30
C for
for
cells 48
water)
hr
were was
at
and
(pH 7.0) Colonies
30
20
This
spread C.
treated
with
purified one
mutant
iodine/potassium white haloes examined. 6 •~
cells
103
at
the
covered
The surviving
of
a
was
portions
of times
cephalosporithe
culture in
was
Table
1.
survivors.
The
0.5% with in
(8 mm/60 mm) cephalosporinase
frequency
nm
to
indicated
benzylpenicillin
iodide indicative
610
culture
as described
10%
and
incubation
and of
A
growth
containing
were
1%
at
C. broth
broth
inducer,
about agar
colonies of
the
density
yield
a solution
per
to and
activity
the
infusion
resulting
and
of
optical
disrupting
heart
added
enzyme
procedure
on
The
containing developing
picked, about
min.
were
The
after
was
removed
20
temperature.
culture
mid-exponential
were of
activity.
the
the
addition
culture
measurement
nase
nized
of
(in
fresh same
of 400 ƒÊg/ml
After
induced
the
635
by ben-
at
a nutrient
with
at
density 0.5
concentration
continued.
in
10-fold
benzylpenicillin
final
growing
overnight
shaking
about
phase),
at
diluted
synthesis
GN346
grown
with
When
for
C. freundii
culture
incubated
IN C. FREUNDII
of cephalosporinase
in
bacterial at
SYNTHESIS
of
starch soft-agar
0.067 at
appearance
NG-mutageand
incubated
(0.5%
M phosphate
agar
room temperature. production at 20 of
the
in
buffer
C
mutant
cells.
The strain GN346/101 behaved as a microconstitutive mutant at 20 C, and increased its enzyme activity with an increase in the growth temperature. When the mutant strain was grown at 30 C, benzylpenicillin, which is the most effective inducer for the enzyme induction in the wild strain, stopped the enzyme synthesis in the mutant strain (Fig.4A). The partially purified enzymes prepared from the cells of GN346/101 grown at 20 C and 30 C were examined concerning their substrate profiles, Km values to ampicillin and cephaloridine, and heat stabilities. No differences were found in these properties between the mutant and parent strains. Mutant strain GN346/16-110 is a constitutive mutant derived from strain GN346/16 and was isolated as follows. The cells of GN346/16 were treated with NG
636
T. SAWAI
Fig.
3.
Induction
of
ET AL
cephalosporinase
synthesis
by
in-
creasing the growth temperature in C.freundii GN346. A bacterial culture grown overnight in nutrient broth at 20 C was diluted 10-fold with prewarmed (20 C) broth, and incubated with shaking at 20 C. When the optical density of the culture at 610 nm reached about 0.4 (in the mid-exponential growth phase), the growth temperature was increased from 20 C to 37 C. Portions of the culture were removed at the indicated times and assayed concerning optical density and specific cephalosporinase activity as described in Table 1. Table 2.
Cephalosporinase activity of mutant strains derived from C. freundii GN346 grown at 20 C or 37 C in the
The
organism
was grown
presence
or absence
in a nutrient
broth
of benzylpenicillin
at the indicated
temperature
in
the presence or absence of the inducer, and assayed for its specific cephalosporinase activity as described in Table 1. The concentrations of the inducer did not have obvious inhibitory effects on the bacterial growth which was followed by measurements of turbidity.
in
a similar
at
20
C for
manner
to
5 hr.
The
containing
200
incubation
at
about
one
,ƒÊg/ml 20
mutant
that
described
above,
mutagenized of
cephaloridine.
C for
two
per
107
days cells
and
cells
were plated.
were The
picked,
cultured spread colonies purified
in fresh on
heart
appearing and
examined.
heart
infusion
infusion on
the
broth
agar plates We
plates after
obtained
CEPHALOSPORINASE
SYNTHESIS
IN
C. FREUNDH
637
A
Fig.
4.
The
relationship
between
B
specific
cephalosporinase
activity
and
the
inducer
(benzylpenicillin) concentration shown by the reverse control type mutants of C. freundii GN 346 grown at various temperatures. The experimental procedures and conditions for the bacterial growth, the induction of cephalosporinase and assay of the enzyme activity are the same as those described in Table 1. The concentrations of benzylpenicillin used did not show obvious inhibitory effects on the normal growth of the organisms. The curves show the results with different initial concentrations of the inducer at a growth temperature of 20c(●
〉, and
30
c(○)。
A,
c. freundii
GN346/101;
B,C.freecndii
GN346/16-110.
The mutant GN346/16-110, as shown in Table 2, is able to produce high cephalosporinase activity without any chemical inducer over a growth temperature range of 20 to 37 C. In Fig. 4B, the enzyme activities of the mutant strain growing in the presence of various concentrations of benzylpenicillin are indicated. Benzylpenicillin acted like a strong corepressor for the synthesis of the enzyme in the mutant at both 20 C and 30 C. No differences in heat stability and other enzymological properties could be detected between the enzyme from GN346/16-110 and the wild enzyme. DISCUSSION
It is known that many cephalosporinases produced by gram-negative rod bacteria are inducible enzymes, and beta-lactam antibiotics act as the inducer. However, little is known about the induction mechanism. Garber and Friedman suggested on the basis of induction kinetics by penicillin that the induction mechanism of cephalosporinase in the pseudomonads is similar to the beta-galactosidase system of Escherichiacoli (4). If the regulatory system for cephalosporinase in C.freundii is analogus to that for lac operon enzymes, the most probable explanation for this unique property of cephalosporinase synthesis may be that the enzyme synthesis is regulated by a highly thermolabile repressor similar to the repressor protein of the its mutant of the lac operon (7,8). The mutant strains GN346/101 and GN346/16-110 are so-called reverse control mutants. The characteristics of the mutants are also similar to those reported for reverse control mutants, the i gene mutants, of the lac operon
638
T. SAWAI
ET AL
(9). Recently, De Graaff et al isolated a C. freundii Hfr donor strain and F-prime factor (3). This has enabled genetical analysis in the species. C. freundii GN346 and its mutants will offer advantages for understanding the molecular mechanism of cephalosporinase induction in gram-negative bacteria. C. freundii GN346 is a clinical isolate and not a specifically isolated mutant for particular experimental purposes in vitro. The thermolabile property of the cephalosporinase synthesis seems to be common in the C. freundii species because other strains of the species, which were randomly selected from our stock strains highly resistant to ampicillin and cephaloridine, possesed this unique property. In our experiments, we could not find a similar regulatory system among the cephalosporinase-producing strains belonging to other species of gram-negative enteric bacteria. We have as yet no information concerning the reason for such a unique regulatory system from the teleological point of view. When C. freundii GN346 was measured for single-cell resistance to cephaloridine and to ampicillin on heart infusion agar plates containing a two-fold dilution of the antibiotics at 37 C, we could not observe any differences in the resistance levels between cells precultured at 20 C and 37 C, respectively. Therefore, the thermolabile regulatory system does not appear to play an important role in bacterial resistance to antibiotics. REFERENCES 1)
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Requests for reprints should be addressed to Dr. Tetsuo Sawai, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Chiba 280, Japan.
