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J Bacteriol, March 1998, p. 1148-1153, Vol. 180, No. 5
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Assembly of Both the Head and Tail of Bacteriophage
Mu Is Blocked in Escherichia coli groEL and
groES Mutants
Régis
Grimaud1,* and
Ariane
Toussaint2
Laboratoire de Génétique des
Procaryotes, Unité Transposition Bactérienne,
Université Libre de Bruxelles, B1640 Rhode St Genèse,
Belgium,2 and
Laboratoire de Microbiologie,
Université Joseph Fourier, F38041 Grenoble Cedex 9, France1
Received 11 November 1996/Accepted 18 December 1997
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ABSTRACT |
Like several other Escherichia coli bacteriophages,
transposable phage Mu does not develop normally in groE
hosts (M. Pato, M. Banerjee, L. Desmet, and A. Toussaint, J. Bacteriol.
169:5504-5509, 1987). We show here that lysates obtained upon
induction of groE Mu lysogens contain free inactive tails
and empty heads. GroEL and GroES are thus essential for the correct
assembly of both Mu heads and Mu tails. Evidence is presented that
groE mutations inhibit processing of the phage head protein
gpH as well as the formation of a 25S complex suspected to be an early
Mu head assembly intermediate.
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INTRODUCTION |
GroEL and its cofactor GroES belong
to a subset of molecular chaperones called chaperonins. They control
the folding of other polypeptide chains, protect them from aggregation,
and regulate the assembly and disassembly of other protein complexes.
Escherichia coli groEL and groES genes are
essential at all temperatures (8). They form an operon which
is constitutively expressed and is induced after heat shock. A 14-mer
of the 57-kDa GroEL protomer associates with a 7-mer of the 10-kDa
GroES protomer to form oligomers which act cooperatively in the
folding of polypeptides. GroEL binds tightly to
nonnative polypeptides. Upon association with GroES, by a
mechanism involving ATP hydrolysis, GroEL discharges the polypeptide in a biologically active conformation (for reviews, see references 6, 17, 18, 29, and
42).
The first groE mutants were identified by their inability to
grow bacteriophage 
or T4. Later, GroEL and GroES were demonstrated to also participate in the lytic cycle of many other bacteriophages. In
all cases, the block caused by groE mutations is in
morphogenesis. However, the steps affected differ from phage to phage.
For and T4, the block is in head assembly (for reviews, see
references 2 and 9), while for T5
and 186, tail assembly is the process requiring GroELS (21,
45).
Several head proteins, including gpB, are cleaved during head
morphogenesis. The defective particles which accumulate in
groEL or groES strains contain only unprocessed
head proteins (19, 20, 22, 37). GroELS was shown to be
necessary for the formation of the preconnector. This small 25S
complex is the first detectable intermediate in head assembly
(34). It consists of 12 subunits of protein gpB, is the
precursor of the head-tail connector, and serves as the initiator for
the assembly of the shell (26, 27, 35).
Like many other phages, Mu does not grow on some groEL and
groES bacteria, although replication, transcription, and
lysis occur normally in such hosts. This finding suggests that GroELS may also be involved in Mu morphogenesis (36). The assembly of Mu virions is under the control of 20 genes arranged in two clusters. The first contains the head genes D, E,
H, I, T, and J; the second
contains the tail genes K, L, M,
Y, N, P, Q, V, W, R, S, and U (13,
14). gpT is the major coat protein. It forms the head shell
(16, 38). gpD and gpE are suspected of being the Mu maturase
components (5a). The protein encoded by gene H
exists in two forms. One, gpH, has a molecular weight which corresponds
to the size predicted from the nucleotide sequence of the H
gene. It is found in a 25S complex which seems to be required for a
very early step in head assembly. The second, gpH*, is found in heads
and is derived from gpH by proteolytic cleavage of its C-terminal end.
gpH processing occurs in assembled heads before DNA packaging
(14).
We have analyzed Mu morphogenesis in groEL and
groES hosts. Our results indicate that both head and tail
assembly are affected. We have traced the main block in head
morphogenesis to a defect in the assembly of the 25S complex and gpH
processing.
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MATERIALS AND METHODS |
Media and general procedures.
Bacteria were grown in LB and
titrated on LA plates containing 1.2% Difco agar (33).
Phage lysates were diluted in SM buffer (40) and titrated on
lawns of sensitive bacteria (0.1 ml of an overnight culture in LB)
poured with 2.5 ml of 0.7% LA agar on LA plates. The phages and
bacterial strains used are listed in Table
1. The purified GroEL and GroES proteins
were gifts from O. Fayet.
In vitro reconstitution.
In vitro reconstitution experiments
were performed as described by Giphart-Gassler et al. (13).
The genotypes of the reconstituted phages were determined by marker
rescue. Plaques of the phage whose genotype was to be tested were
transferred with a toothpick to a mixed lawn of B178 and B178 lysogenic
for one of the two amber mutants used in the in vitro reconstitution.
The plates were incubated at 42°C. These tests showed a clear region
of cell lysis if the reconstituted phage and the phage obtained by
lysogen induction could recombine to yield wild-type phages.
Purification of phage particles.
Phage particles obtained by
thermal induction of lysogens were concentrated by polyethylene glycol
precipitation and purified by ultracentrifugation through a CsCl
gradient, followed by another ultracentrifugation through a sucrose
gradient as described by Grimaud (14). Ultracentrifugation
of total protein extracts was carried out as described previously
(14).
Immunoblotting.
Total protein extracts were prepared as
described by Grimaud (14). Proteins were separated by sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)
(28). Immunoblotting was performed as described by Geuskens
et al. (12) except that electrotransfer was carried out with
a Bio-Rad apparatus for 4 h at 100 V for small gels and overnight
at 50 V for large gels. Anti-gpH* antibody was used at a 1,000-fold
dilution. Anti-GroEL IgG (Epicentre) was used at 0.2 µg/ml.
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RESULTS |
Head-tail assembly is blocked in groEL and
groES hosts.
Mu growth was tested on several
groEL and groES hosts, among which some did and
some did not allow the phage to form plaques (36a). Among
the latter, two groEL strains (groEL44 and
groEL140) and one groES strain
(groES606) (described in Table
2) were chosen for further investigation
of the role of the GroELS chaperonin in Mu morphogenesis. These strains
were lysogenized with Mucts62pAp1 and induced at 42°C.
Table 3 shows that under conditions where a wild-type lysogen produces lysates containing 2 × 109 phages/ml (data not shown), phage production from the
groE lysogens was severely reduced, the number of
plaque-forming phages varying from 2 × 105 to 6 × 106 phages/ml (i.e., 10
4 to 0.02 phage/bacterium).
In experiments where infectious particles were reconstituted in vitro
by mixing a lysate produced by a head gene mutant (i.e.,
tail donor)
with one produced by a tail gene mutant (i.e., head
donor),
Giphart-Gassler et al. (
13) have shown that like other
tailed bacteriophages, Mu assembles its heads and tails separately.
These then join to form complete infectious virions. We used the
same
in vitro reconstitution assay to see which process or processes,
i.e.,
head assembly, tail assembly, or both, is (are) deficient
in
groE strains. Mu
cts62pAp1 lysates were prepared
on each of
several
groEL and
groES hosts
(
groE lysates). They were mixed
with either a tail or a head
donor lysate obtained by growing
head mutant
Mu
cts62
Tam1913 or tail mutant
Mu
cts62
Lam1007 on a
Sup
0 strain. The
results, summarized in Table
3, show that when either
heads or tails
were added to a
groE lysate, the number of plaque-forming
phages always increased (by a factor of 2, to over 100). Always,
however, such phages remained 10 to over 100 times less abundant
than
in control experiments where the head and tail donor lysates
were mixed
together. To test whether the
groE lysates contain
some
inhibitory factor preventing the normal joining of fully
functional
heads and tails, we grew Mu
cts62pAp1 on the
groEL140 host and mixed the resulting lysate with both head
and tail donor
lysates. The level of reconstitution was exactly the
same as in
the control experiment where no
groE lysate was
added (Table
3).
The simplest interpretation of the reduced
reconstituted phage
yields obtained with
groE lysates and
heads or tails is that GroEL
and GroES are required for both head and
tail morphogenesis.
Reconstituted phages have the genotype of the head donor. We took
advantage of this property to identify the true head donor
in
reconstitution experiments where a head donor lysate was mixed
with
groE lysates. Phages whose heads come from the
groE lysate
should be Am
+; those whose heads
come from the head donor lysate should be
amber. Table
3 shows that
there were both amber and Am
+ particles among the
reconstituted phages, the latter representing
9 to 76% of
the reconstituted population. The presence of reconstituted
amber phages indicated that the
groE lysates contained
some free
active tails. The production of Am
+ phages
suggested that
groE lysates contained unstable or/and
incomplete and hence noninfectious particles with a
Mu
cts62pAp1
genome that were rescued upon addition of a head
donor lysate.
Rescue could result from the addition of one or more
factors present
in the added lysate and coming from either the phage
(e.g., proteins
such as accessory proteins involved in the
stabilization of the
capsid, tail fiber proteins which are normally
added after head-tail
joining) or the bacterial host (e.g., GroEL and
GroES). We tested
the tail fiber hypothesis by looking for production
of Am
+ particles after adding heads produced by a
Mu
Sam tail fiber mutant
(
15) to a
groE
lysate. Am
+ phages were as abundant as with other head or
tail donor lysates
(data not shown). This ruled out an involvement of
tail fiber
proteins. We also tested the possibility that
Am
+ phage production resulted from the addition of GroELS
present
in the head donor lysate. Addition of purified GroEL and/or
GroES
to a
groE lysate did not increase the formation of
Am
+ phages (data not shown).
We next attempted to test head and tail morphogenesis
separately in
groE hosts. Three tail mutants
(Mu
cts62
Lam1007,
Mu
cts62
Nam1041,
and
Mu
cts62
Yam1027) and three head mutants
(Mu
cts62
Ham7100,
Mu
cts62
Iam4037,
and Mu
cts62
Tam1913) were grown on the
groEL140 strain and on
the
groES619 strain. Each resulting lysate was mixed
with either
a head donor (
Lam1007) or a tail donor
(
Tam1913) lysate grown
on a Sup
0
GroE
+ host. Table
4 shows
that each head mutant grown on a
groE host
supplied tails as
well as the control tail donor lysate did. In
the reverse case, heads
provided by a tail mutant grown on
groE allowed for 2- to
20-fold less efficient reconstitution compared
to the control head
donor lysate. Thus active tails were produced
in the
groE
host provided head assembly was blocked, but head
assembly remained
partially blocked in the absence of tail assembly.
groE lysogens produce free tails and empty heads.
To further characterize the defect in Mu morphogenesis, we
purified, on CsCl and sucrose gradients, the particles present in
Mucts62pAp1 lysates obtained after thermal induction
of groEL or groES lysogens (see Materials
and Methods for the detailed protocol). Only particles with a 1.3-g/ml
density, i.e., tails and/or empty heads (16), were detected.
Sucrose gradient fractions were analyzed by SDS-PAGE, which enabled us
to further distinguish defective heads (identified by the presence of
gpT, the major head protein) from tail-related particles (identified by
the presence of gpL, the major tail protein). All lysates contained
head-related particles with a sedimentation coefficient of 100S, i.e.,
particles sedimenting like empty heads. Tail-related particles with a
normal 90S sedimentation coefficient were also detected (Fig.
1A) (14).
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FIG. 1.
Protein composition of Mu particles produced on
groE hosts. (A) Sedimentation profiles of Mu particles
produced by
B178groEL140(Mucts62pAp1).
Phage particles were isolated on CsCl gradients at a 1.3-g/ml
density and run on a 10 to 50% (wt/wt) sucrose gradient at 45,000 rpm
for 60 min at 5°C in a Beckman SW50.1 rotor. The sedimentation
coefficients were estimated as described in reference
32.  , relative percentage of gpT;
---, relative percentage of gpL. The relative amounts
of gpT and gpL in each fraction were determined by scanning Coomassie
blue-stained SDS-polyacrylamide gels (see Materials and Methods for
details). Results for B178groES606
(Mucts62pAp1) and
B178groEL44(Mucts62pAp1) were the same as for
B178groEL140(Mucts62pAp1) and are therefore not
shown. Sedimentation is from left to right. (B) Fractions containing
the head peaks as identified in panel A were analyzed by SDS-PAGE
(12.5% gel). The faint bands around 60 kDa were not present in all
preparations and were thus probably contaminants. Lane 1, head peak
fraction (no. 10) produced by B178groEL44
(Mucts62pAp1); lane 2, head peak fraction (no.
10) produced by B178groEL140(Mucts62pAp1);
lane 3, head peak fraction (no. 10) produced by
B178groES606(Mucts62pAp1).
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The protein composition of the particles present in the head peak
fractions of the sucrose gradients was investigated by SDS-PAGE
(Fig.
1B). Empty heads and tails had very similar sedimentation
coefficients
and did not separate well. The head peak fractions
displayed gpL and
two tail proteins (average molecular size, 38
kDa) (Fig.
1B)
(
13) in addition to gpT. The protein composition
of the
tails present in
groE lysates was thus no different from
that of tails in wild-type lysates. gpT was the only head protein
present in the
groE lysates. Previous analysis
(
14) showed that
complete Mu heads and most Mu empty heads
contain both gpT and
gpH*, a processed form of gpH. The empty heads
produced in
groE hosts showed no evidence of any gene
H product (Fig.
1B). This
was confirmed by immunoblotting
analysis with anti-gpH* antibody
of the same gradient fractions (data
not shown).
To test whether the absence of gene
H products in
groE heads was due to a defect in gpH synthesis or gpH
incorporation into
the head, we probed total proteins obtained from
induced
groE lysogens with anti-gpH* antibody. gpH was
detected in all extracts,
whether derived from
groEL or from
groES strains lysogenic for
Mu
cts62pAp1 (Fig.
2). However, the processed form gpH* was
always
much less abundant in
groE extracts.
B178
groEL140(Mu
cts62pAp1)
(Fig.
2, lane 3) and
B178
groEL44(Mu
cts62pAp1) (Fig.
2, lane 2)
displayed a small amount of gpH*, while in
B178
groES606(Mu
cts62pAp1)
(Fig.
2, lane 1), gpH*
was not detectable. Synthesis of gpH was
thus normal in
groE
hosts, but gpH processing and incorporation
into the head did not
proceed correctly.
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FIG. 2.
Immunoblotting analysis of proteins synthesized in
groE lysogens. Proteins were extracted from induced lysogens
and separated on denaturing gels (12.5% acrylamide). The gels were
probed with anti-gpH* as described in Materials and Methods. About 8 µg of protein was loaded in each lane. Lanes: 1, B178groES606(Mucts62pAp1); 2, B178groEL44(Mucts62pAp1); 3, B178groEL140(Mucts62pAp1); 4, B178(Mucts62pAp1); 5, B178.
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The
groEL44 mutant has one mutation in the
35 region of
the
70 promoter of the
groELS operon (Table
2). This mutation should
play no major role in the phenotype of this
mutant, as expression
of the
groE operon is mostly under the
control of the
32 promoter at temperatures between 30 and 43°C (
44). In addition,
groEL44
thermoresistant revertants which retained the promoter
mutation were
isolated (
43). To check that in our experiments
expression
of the
groELS operon was not reduced by the promoter
mutation, we used immunoblotting with anti-GroEL antibody to determine
the amount of GroEL protein in our
groEL44-derived strains.
It
was the same as that of GroEL in wild-type strains (data not shown).
groE mutations block an early step of Mu head
assembly.
gpH was shown to be part of a 25S complex appearing as a
likely very early intermediate in Mu head assembly (14). We
looked for the presence of this complex in wild-type and
groE strains lysogenic for Mucts62pAp1. Crude
extracts of the induced lysogens were loaded on sucrose gradients.
After centrifugation, the gradients were fractionated and fractions
were analyzed by immunoblotting with anti-gpH* antibody. In wild-type
extracts (Fig. 3A), gpH migrated to the
top of the gradient with unassembled materials (fraction 1) and to the
position of the 25S complex (fractions 9 and 10). gpH* was found only
at the bottom of the gradient (fraction 20). Fraction 20 was previously
shown to contain also the Mu coat protein gpT. Most likely, only
complete virions and head-related particles with a high sedimentation
velocity migrate to that position (14). The situation in
groE extracts was clearly different. gpH was present only in
the top fractions (fraction 1) of the gradient (see Fig. 3B),
indicating that in the groE strains, gpH incorporation into
the 25S complex does not proceed normally. Very low amounts of gpH*
could be seen in some groE extracts (Fig. 2), which led us
to check the sucrose gradient fractions for the presence of the gpH
truncated form. Although a low amount of gpH* sometimes appeared in the
top fractions, despite several attempts, it could never be detected in
the bottom of the gradients (data not shown). This finding suggested
that in groE strains, gpH* was not associated with
head-related structures and hence that gpH was not cleaved along the
proper assembly pathway.
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FIG. 3.
Sedimentation analysis of particles present in induced
groE lysogens. Total protein extracts prepared after
induction of lysogens were run on a 10 to 40% (wt/wt) sucrose gradient
at 50,000 rpm for 3 h at 5°C in a Beckman SW50.1 rotor. The
gradients were fractionated from top to bottom, and the fractions were
analyzed by immunoblotting with anti-gpH*. (A)
B178(Mucts62pAp1) extract; (B)
B178groEL140(Mucts62pAp1) extract. Sedimentation
is from left to right. The cross-reacting band present in fractions 2 and 3 was previously observed in extracts prepared from nonlysogenic
bacteria grown at 32°C and shifted to 42°C. The other
cross-reacting band present in fraction 8 was identified as GroEL by
immunoblotting with anti-GroEL antibodies (data not shown).
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DISCUSSION |
With Mu, both head and tail assembly are affected in
groE hosts.
Our in vitro reconstitution experiments
(Tables 3 and 4) show that only few active heads and tails are produced
when Mu multiplies in groEL or groES hosts,
suggesting that the GroELS chaperonin is required for the correct
assembly of Mu heads and tails. This contrasts with observations on
other phages where either head or tail assembly is affected (head
assembly for , T4, and HK97; tail assembly for 186 and T5) (2,
5, 9, 21, 45).
and T4 mutants which have recovered the ability to form plaques on
groE hosts can be readily isolated at frequencies ranging
from 10
6 to 10
7 (
10,
11). Yet,
despite the use of various mutagens and several
different
groEL and
groES alleles, we failed to find
similar Mu
mutants. This result is consistent with both Mu head and Mu
tail
assembly being blocked in
groE hosts, as more than one
mutation
in Mu might then be required to overcome the
groE
defect, and
double mutants might be too rare to be detected.
The major effect of groE mutation on Mu head assembly
is at the level of the Mu head protein gpH.
In Mu lysates prepared
on groE hosts, we identified empty heads and inactive free
tails. The defect in tails remains to be elucidated since they appeared
unchanged in all of our analyses. The presence of empty heads suggests
that head assembly is blocked before DNA packaging. Contrary to what
happens in a GroE+ host, the head protein gpH, although
produced in a normal amount, does not assemble into a 25S complex, is
not efficiently processed into its cleaved gpH* form, and is not
incorporated into heads. This assembly defect is similar to that
observed with MuHam mutants such as MuHam7100
which express no gpH and do not assemble the 25S complex (14,
16). Since this complex seems to be an early head assembly
intermediate (14), blocking its formation would block gpH
incorporation in the head and hence gpH processing which occurs only in
assembled heads. groE mutations thus appear to cause a major
and specific block in head assembly at the level(s) which involves gpH.
The role of GroELS in phage head assembly has been studied in great
detail with phages HK97 and
. The HK97 coat protein gp5
aggregates
in
groE hosts. A complex between GroEL and gp5 was
isolated
and used in vitro to show that GroELS promotes gp5 folding
(
5,
41). During
head assembly, GroELS specifically interacts
with
gpB. Mutations bypassing the
groE mutations have been found
in gene
B, and a biologically active GroEL-gpB complex has
been
identified (
10,
11,
39). In these two cases,
groE mutations
cause a specific defect because the
chaperonin is required to
activate one particular head protein. The
situation for Mu head
appears similar since our results suggest that Mu
gpH requires
GroELS to assume its functional state. In
groE
hosts, because
of delayed or incorrect folding, gpH would not be
available for
head assembly, and hence the resulting defect appears
similar
to that observed with mutant Mu phages which express no gpH.
It was proposed earlier that gpH could be a functional homolog of the
gpB portal protein (
14). The results cited above
support
that hypothesis and the similarities that exist between
Mu and
head
assembly pathways.
How groE mutations can affect the production of
native polypeptides and block Mu assembly.
Mu
lysates grown on groE hosts, although containing empty heads
similar to those produced by MuHam mutants, also contain
large amounts of defective free tails and smaller quantities of
infectious phages, active free tails, and particles which become active
upon combination with either head or tail donor lysates. In
MuHam mutant lysates, in vitro reconstitution experiments
did not provide any evidence for the existence of other types of
phage-related particles besides empty heads (our unpublished results).
The effect of groE mutations on Mu assembly thus cannot
result from the sole absence of active gpH. The diverse defective
particles produced, rather, reflect a requirement for GroELS at several
morphogenetic steps or a deleterious effect of inactive gpH on several
assembly steps. A direct effect of groE mutation on several
morphogenetic steps implies that the chaperonin is required not only
for the folding of gpH. This view is perfectly compatible with the
properties of GroELS. Chaperonins play a general role in protein
folding and seem to be required for the folding of many
polypeptides. Horwich et al. (23) found that in
bacteria lacking GroEL activity, 30% of the newly synthesized proteins
aggregate. More recently, the flux of newly synthesized
polypeptides through the chaperonin has been investigated.
Under nonstress conditions, 10 to 15% of all newly synthesized
polypeptides interact with GroEL (7). It is thus
very likely that several Mu morphogenetic proteins require GroELS to
reach their native state.
Among the different Mu-related particles produced in
groE
strains, the minor defective types likely derive from intermediates
which escaped the major early defects in assembly of the 25S complex.
These less abundant defective particles might also reflect a weaker
dependence on GroELS of other phage proteins required for later
assembly steps.
The
groE mutants that we used in our experiments were
characterized in great detail. Zeilstra-Ryalls and coworkers
(
43)
showed that
groEL140 and
groEL44
strains which are thermosensitive
for growth will grow at the
nonpermissive temperature provided
that the mutant proteins are
overproduced. This finding suggests
that the mutations, rather than
knocking out the chaperonin's
ability to fold the substrate protein
correctly, decrease the
folding rate. Biochemical analysis of the
GroEL140 protein confirmed
that it still binds substrate
polypeptides normally but releases
them abnormally slowly
(
1). Phage morphogenesis is known to
require the assembly,
in the right sequence, of a controlled amount
of intermediates
(
4). If in
groE strains the folding of some
morphogenetic polypeptides is slowed down, the normal progress
of the assembly steps will be disrupted. In phage
,
mutants,
some of which carry a mutation in gene
E, overcome the
groE defect
(
11,
39). To account for this
observation, it was proposed
that in
groE hosts, assembly of
phage particles aborts because
the slower release of the active form of
one component disrupts
the balance between this component and other
morphogenetic proteins.
By decreasing the rate of synthesis of gpE, the
mutation would
restore the balance between the slowly released
component and
gpE and hence restore assembly (
11,
39). A
similar process
could account for the partial restoration of head
assembly and
total restoration of tail morphogenesis which we observed
upon
growing Mu tail or head mutants in a
groE host. Very
large quantities
of late phage proteins are produced during the lytic
cycle. In
groE mutants, delayed release of GroEL-bound
peptides could limit
the amount of chaperonin available and hence the
production of
morphogenetic proteins competent for assembly. If
production of
either head or tail components is blocked (e.g., by an
am mutation),
more chaperonin would become available for
those assembly steps
which heavily rely on it.
Recently, the dependence on the chaperonin for folding of newly
synthesized proteins has been investigated in
E. coli
(
7).
Three classes of proteins were distinguished: (i) a
minor class
consists of mostly small polypeptides which do not
bind to GroEL;
(ii) a second class includes the majority of the
proteins which
are largely independent of the chaperonin although about
5% of
each of them bind to GroEL; and (iii) 10% of the newly
synthesized
polypeptides with sizes ranging between 25 and 55 kDa are strictly
dependent on GroEL for their folding. In our study,
the Mu gpH
protein appears to strongly depend on GroELS for function
and
hence would belong to the third class. Other Mu morphogenetic
proteins seem to be affected by
groE mutations to a lesser
extent.
These could belong to the second class, and only a small
fraction
of them would rely on GroEL for folding. In
groE
mutants, most
gpH would be nonfunctional, leading to the accumulation
of defective
heads. Few functional assembly intermediates could be
formed despite
that primary block and proceed to further assembly steps
which,
depending on whether they do or do not rely on proteins
belonging
to the second class, will or will not proceed normally in the
groE strain. This could lead to the formation of the minor
types
of defective particles that we observed. Our results are thus
consistent with the view that the role of GroELS in Mu assembly
mimics the general role of the chaperonin in the host bacterial
cell.
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ACKNOWLEDGMENTS |
We thank C. Georgopoulos for helpful discussions and for
providing bacterial strains, O. Fayet for the gift of GroE proteins, and M. Pato for his contribution to the initial part of this work.
This work was carried out with support from the Fonds National de la
Recherche Scientifique and the Brachet Stiftung. R.G. was a fellow of
the Ministère de la Recherche et de l'Enseignement Supérieur (France); A.T. is a fellow of the Directeur de
Recherche from the Fonds National de la Recherche Scientifique
(Belgium).
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FOOTNOTES |
*
Corresponding author. Present address: Laboratory of
Cell Biology, Building 37, Room 1B09, National Cancer Institute,
Bethesda, MD 20892. Phone: (301) 435-1938. Fax: (301) 402-0450. E-mail: regis{at}helix.nih.gov.
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J Bacteriol, March 1998, p. 1148-1153, Vol. 180, No. 5
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
