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Journal of Bacteriology, May 1999, p. 2970-2972, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Disulfide Bridges Are Not Involved in
Penicillin-Binding Protein 1b Dimerization in Escherichia
coli
Christian
Chalut,1
Marie-Hélène
Remy,1 and
Jean-Michel
Masson1,2,*
Institut de Pharmacologie et de Biologie
Structurale du CNRS1 and Institut
National des Sciences Appliquées de
Toulouse,2 Toulouse, France
Received 8 December 1998/Accepted 24 February 1999
 |
ABSTRACT |
PBP1b can be found as a dimer in Escherichia coli.
Previous results suggested that dimerization involved the cysteine(s)
in an intermolecular disulfide bond. We show that either deletion mutants or a mutant without cysteines is fully active and still binds
penicillin and that the latter can also form dimers.
| |
TEXT |
Penicillin-binding proteins (PBPs)
are a set of membrane-bound enzymes involved in the late stages of
peptidoglycan biosynthesis (for reviews, see references
4 and 13). PBPs in connection with several other enzymes, including hydrolases and synthetases, are
essential in the control of bacterial morphology, elongation, and cell
division (2, 3, 10) and must have activities coordinated in
both time and space. Cell division is directed by a multimeric
structure, the divisome (12). Recent findings established
that some PBPs belong to this enzymatic complex and take part in the
septum formation (3, 11). Moreover, it has been shown that
some PBPs interact with each other and with lytic transglycosylases,
suggesting the existence of murein-synthesizing enzyme complexes
(14, 19). However, the composition and the functions of
these complexes remain speculative and require further investigation.
PBP1b is the major enzyme for peptidoglycan synthesis in
Escherichia coli (18) and should thus play a
prominent part in these complexes. This transmembrane protein is
organized into two catalytic domains harboring transpeptidase and
transglycosylase activities (8, 16). It is usually present
in the cell membrane in three molecular forms, termed 
, 
, and
, with slightly different electrophoretic mobilities
(15). The component derives from PBP1b by proteolytic
release of the 24 N-terminal residues, whereas the component arises
from a translation start site downstream from the PBP1b translation
start site of the ponB gene (17). Earlier studies
have established that PBP1b can occur as dimers which can be detected
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
at a position corresponding to a molecular mass of about 140 kDa
(23, 24). Two-dimensional gel analysis confirmed that this
high-molecular-weight band consists of two PBP1b monomers
(23). Two classes of dimers have been characterized as
having different biochemical properties, but both need
-mercaptoethanol for dissociation, suggesting a stabilization by
intermolecular disulfide bonds involving one or both cysteines of PBP1b
(C776 or C794), located at the carboxy-terminal end of the protein. In
this study, we confirmed that PBP1b can exist in a stable dimeric form.
However, we also show that disulfide bridges are dispensable for PBP1b
activities in vitro and in vivo and that PBP1b mutants without cysteine
residues are still able to dimerize.
PBP1b dimers can dissociate under mild denaturation or
solubilization conditions.
PBP1b was produced in strain QCB1,
which was transformed with vector pPONB (8). QCB1 is an
E. coli strain with a deletion in gene ponB,
which codes for PBP1b (1). Plasmid pPONB expresses PBP1b
with a tag peptide (YPYDVPDYA) from the hemagglutinin HA1 epitope of
influenza virus (21) at its C-terminal end. The expressed PBP1b can be visualized by Western blotting, performed on E. coli membrane preparations with monoclonal antibody 12CA5 raised
against the tag peptide, as described by Lefèvre et al.
(8).
Membrane fractions (8) from QCB1(pPONB) cells grown at
37°C were incubated for 10 min in sample buffer (7) with
or without -mercaptoethanol, at either room temperature or 100°C,
and applied to SDS-PAGE gels for Western blot analysis. Three major
bands were detected at positions corresponding to the three molecular forms of PBP1b commonly found by SDS-PAGE (PBP1b, -, and -) (Fig. 1, lanes 2 through 5). With samples
incubated at room temperature, an additional band reacted with the
antitag antibody at about 150 kDa (Fig. 1, lanes 4 and 5),
corresponding to the previously described dimeric form of PBP1b
(23, 24). This band was never detected with boiled samples
(Fig. 1, lanes 2 and 3). Furthermore, incubation of samples containing
-mercaptoethanol at several temperatures for 10 min showed that the
dimeric form is stable at 60°C but almost totally dissociated at
80°C (Fig. 1, lanes 6 through 9). We observed the same pattern of
dissociation in samples without -mercaptoethanol (data not shown).
These results indicate that dissociation of the dimer occurs when
temperature increases, with or without -mercaptoethanol, thus
suggesting that disulfide bonds do not take part in the stabilization
of the dimer.
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FIG. 1.
PBP1b dimer stability. Membrane fractions prepared from
strain QCB1 (lane 1) or QCB1(pPONB) (lanes 2 through 10) were incubated
for 10 min in sample buffer with 5% or no -mercaptoethanol (-Me)
at temperatures ranging from room temperature (RT) to 100°C. Lane 10*
shows a soluble membrane fraction after treatment with Nonidet
P-40-NaCl (see text). Samples were submitted to SDS-PAGE
(7). Western blotting with the antitag 12CA5 antibody was
performed with the Enhanced Chemiluminescence detection system
(Amersham). M, monomeric forms of PBP1b; D, dimeric forms of PBP1b;
temp., temperature, in degrees Celsius.
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|
In an attempt to solubilize the dimeric form of PBP1b in a native
conformation, membranes were incubated with 0.5% Nonidet
P-40-0.5 M
NaCl for 30 min at 25°C and centrifuged for 30 min
(14,000 ×
g, 4°C). Most of the PBP1b was recovered in the soluble
fraction. This solubilized PBP1b was still able to bind fluorescein
6-amino-penicilloic acid (6-APA-Flu) (
8), indicating a
correct
fold (data not shown). The solubilized fraction was incubated
for 10 min at room temperature in sample buffer without
-mercaptoethanol
prior to SDS-PAGE and Western blot analysis with
the antitag antibody.
Under these conditions, we failed to detect the
dimeric form of
the protein (Fig.
1, lane 10*). Since the mild
solubilization
conditions used in this experiment are unlikely to break
a covalent
bond, these observations give further support to the idea
that
PBP1b dimers are not associated by a disulfide
bond.
PBP1b mutants without cysteine are active in vitro and in
vivo.
To further investigate the role of a possible disulfide
bridge in PBP1b activities, we constructed several mutants by the inverse-PCR technique (20). These constructs were checked by DNA sequencing. pV775
Ct and pL793Ct mutants carry deletions of
the sequence corresponding to the C-terminal region of PBP1b. They were
made by fusing the V775 codon of pV775Ct or the L793 codon of
pL793Ct directly to the tag sequence. The truncated protein produced
by plasmid pL793Ct still contains one cysteine residue (C776),
whereas the one which is produced by pV775Ct does not. A third
mutant (the pCys
mutant) was made by replacement of the
C776 and C794 codons by alanine codons. These constructs were
transformed in the QCB1 strain, and activities of the resulting PBP1b
mutants were examined.
Membrane fractions were prepared from QCB1 harboring the mutated
plasmids. Western blot analysis with antitag antibody 12CA5
revealed
that the three PBP1b mutants are produced at the same
level as the
wild-type protein in bacterial cells (Fig.
2A, lanes
2 through 5). The three PBP1b
mutants also bind fluorescent penicillin
as efficiently as the wild
type, indicating a proper folding and
a functional transpeptidase
activity (Fig.
2B, lanes 2 through
5).
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FIG. 2.
PBP1b mutants. (A) Production of the PBP1b mutants.
Membrane fractions from QCB1 (lane 1) or QCB1 transformed with pPONB
(lane 2), pCys (lane 3), pL793Ct (lane 4), and
pV775Ct (lane 5) were analyzed by Western blotting with the antitag
12CA5 antibody. (B) Binding of 6-APA-Flu by the PBP1b mutants. Membrane
fractions from QCB1 (lane 1) or QCB1 transformed with pPONB (lane 2),
pCys (lane 3), pL793Ct (lane 4), and pV775Ct (lane
5) were incubated with 6-APA-Flu and analyzed by Western blotting with
an antifluorescein antibody. 1a, PBP1a; 1b, PBP1b.
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|
The ability of these mutants to restore the PBP1b activities in the
synthesis of the peptidoglycan was tested by the disc
agar diffusion
method (
8,
9). Growth inhibition diameters
were also
measured on agar plates with 0.3 µg of cephaloridine
per ml. Under
these conditions, PBP1a is specifically inactivated
and cell growth
thus relies solely on PBP1b (
22). QCB1 harboring
the mutated
plasmids exhibits the same antibiogram patterns as
the wild-type strain
MC6-RP1 or QCB1 harboring pPONB (Table
1).
The PBP1b mutant resulting from the
expression of pCys
is fully active in vivo, indicating
that replacement of the two
cysteine residues by alanine does not
affect peptidoglycan synthesis.
This result demonstrates that dimeric
forms of PBP1b stabilized
by a disulfide bridge are not required for
peptidoglycan synthesis.
The mutant resulting from the expression of
pL793
Ct in strain
QCB1 is as active as the wild-type PBP1b,
confirming that the
last 51 amino acids of PBP1b are not required for
in vivo activity.
This result is not surprising, since it has been
shown that the
C-terminal residues down to M780 are dispensable for the
in vitro
or in vivo activity of PBP1b (
5,
6). Interestingly,
the
pV775
Ct mutant in strain QCB1 is slightly less active than the
wild type. It has been shown that the deletion of the C-terminal
domain
of PBP1b down to D762 yields a mutant that is inactive
in vitro and in
vivo (
8). Combined with our previous alanine
stretch
scanning results (
8), our present results strongly
suggest a
crucial role in PBP1b structure for residues 766 through
772.
pCys PBP1b is able to form dimers.
Membrane
fractions of strain QCB1 harboring pCys were incubated in
sample buffer, with or without -mercaptoethanol, either at room
temperature or 100°C for 10 min, and analyzed by Western blotting
with the antitag antibody 12CA5. Results clearly show that this mutant
can dimerize and dissociate under the same experimental conditions as
the wild-type protein (Fig. 3, lanes 2 through 5). Furthermore, the stability of the dimeric form of the PBP1b
mutant expressed from pCys was tested at several
temperatures. The dimer remains stable after incubation for 10 min at
60°C with sample buffer containing -mercaptoethanol but is almost
totally dissociated at 80°C. The pattern of dissociation was similar
when the experiment was performed with sample buffer without
-mercaptoethanol (data not shown). Both deletion mutants are also
able to form dimers (not shown).
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FIG. 3.
Dimers of the pCys PBP1b mutant. Membrane
fractions prepared from strain QCB1 (lane 1) or
QCB1(pCys) (lanes 2 through 5) were incubated with sample
buffer with 5% or no -mercaptoethanol (-Me) at room temperature
(RT) or 100°C for Western blotting analysis with antitag 12CA5
antibody. Abbreviations are the same as in Fig. 1.
|
|
Zijderveld et al. (
23,
24) have shown that PBP1b exists as a
dimer in
E. coli. These authors characterized two classes
of
dimers, one bound to the inner membrane and a second associated
with
the peptidoglycan. Our results show that although the PBP1b
dimers are
fairly stable, being detected in SDS-PAGE and requiring
a 10-min
incubation at 80°C to be dissociated, the dimerization
does not rely
on the formation of disulfide bonds as previously
hypothesized.
Moreover, we demonstrated that the two cysteine
residues of PBP1b are
dispensable for both its activity in vivo
and its dimerization. Mild
solubilization with detergents suffices
to dissociate the dimers, but
we failed to detect any effect of
zinc on the dimer stability as was
previously shown (
24). This
discrepancy may result from
differences in the preparation of
membranes. PBP1b consists of three
components (
,
, and
) with
different mobilities on SDS-PAGE
gels. Two-dimensional gel analysis
suggests that only homodimers,
-
,
-
, and
-
, occur (
23).
We have not
observed these different forms, perhaps due to their
close migration in
SDS-PAGE. We noticed, however, that the 150-kDa
band sometimes appeared
as a doublet. Whether this doublet corresponds
to two classes of dimers
is under
investigation.
The biological role of these dimers still remains to be experimentally
demonstrated, although the recent models of peptidoglycan
synthesis
imply that PBP1b acts as a dimer within a multienzymatic
complex
(
4,
13). The above-described experiments exclude
models in
which PBP1b is linked by disulfide bridges. Nevertheless,
these dimers
involve strong noncovalent interactions. Further
investigations are
necessary to elucidate the precise nature of
these interactions and
their roles in PBP1b
activities.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: IPBS-CNRS,
205 Route de Narbonne, 31077 Toulouse Cedex, France. Phone: (33)
0561.17.54.76. Fax: (33) 0561.17.59.94. E-mail:
masson{at}ipbs.fr.
| |
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Journal of Bacteriology, May 1999, p. 2970-2972, Vol. 181, No. 9
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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