J Bacteriol, April 1998, p. 1632-1641, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo 05599-970, Brazil
Received 28 October 1997/Accepted 2 February 1998
| |
ABSTRACT |
|---|
|
|
|---|
The groESL operon is under complex regulation in
Caulobacter crescentus. In addition to strong induction
after exposure to heat shock, under physiological growth conditions,
its expression is subject to cell cycle control. Transcription and
translation of the groE genes occur primarily in
predivisional cells, with very low levels of expression in stalked
cells. The regulatory region of groESL contains both a
32-like promoter and a CIRCE element. Overexpression of
C. crescentus
32 gives rise to higher levels
of GroEL and increased levels of the groESL transcript
coming from the
32-like promoter. Site-directed
mutagenesis in CIRCE has indicated a negative role for this
cis-acting element in the expression of groESL
only at normal growth temperatures, with a minor effect on heat shock
induction. Furthermore, groESL-lacZ transcription fusions
carrying mutations in CIRCE are no longer cell cycle regulated. Analysis of an hrcA null strain, carrying a disruption in
the gene encoding the putative repressor that binds to the CIRCE
element, shows constitutive synthesis of GroEL throughout the
Caulobacter cell cycle. These results indicate a negative
role for the hrcA gene product and the CIRCE element in the
temporal control of the groESL operon.
| |
INTRODUCTION |
|---|
|
|
|---|
The heat shock response is a universal phenomenon by which all living cells, when exposed to temperatures higher than their normal physiological temperatures, induce the synthesis of a group of proteins generally known as the heat shock proteins (Hsps).
In prokaryotes, the best-studied mechanism of heat shock induction is
that of the gram-negative bacterium Escherichia coli. In
this organism, the heat shock response is positively regulated at the
level of transcription by the alternate sigma factor
32,
encoded by the rpoH gene, whose levels increase drastically (about 20-fold) during the first few minutes after heat shock due to
derepression of its translation and to a transient increase in its
half-life (for a review, see reference 32). The
level and activity of
32 are negatively regulated in
E. coli by the products of the heat shock genes
dnaK, dnaJ, and grpE. DnaK, DnaJ, and
GrpE cooperate to present
32 to the FtsH protease, which
degrades the heat shock sigma factor (14, 26), whereas DnaK
and DnaJ work together to inhibit
32 activity in heat
shock gene transcription by preventing its binding to the RNA
polymerase core and GrpE partially reverses this inhibition (9).
In contrast to E. coli, no evidence for involvement of
32-like factors in heat shock gene expression was found
in gram-positive bacteria, such as Bacillus subtilis
(13) and Clostridium acetobutylicum (19). However, a highly conserved inverted repeat (IR)
sequence was detected in front of some of the major heat shock genes
(dnaK and groE), which was proposed to substitute
for the
32 regulation of the heat shock response in
gram-positive bacteria. It soon became apparent that this inverted
repeat sequence, also known as the CIRCE element (named CIRCE for
controlling inverted repeat of chaperone expression), was more
widespread than initially imagined, being identified in front of the
groESL operons of several gram-negative bacteria, including
those presenting
32-like promoters (2, 24).
The role of the CIRCE element has been investigated, and evidence indicates that it functions as an operator site to which a repressor binds (24, 31, 33). The protein coded by orf39, the first gene in the dnaK operon of B. subtilis, with homologs found in several other bacteria and recently renamed hrcA (22, 23), was found to bind the IR at the DNA level and to serve as the repressor in B. subtilis (31).
In the gram-negative bacterium Caulobacter crescentus,
several heat shock-inducible genes have been characterized and they all
present
32-like promoters (2, 11, 22). The
gene encoding the
32 homolog of C. crescentus
has been isolated, and one of its promoters (P2) aligns with the
32 consensus sequence, with transcription from this
promoter increasing dramatically during heat shock (21, 29).
Recently, in vitro transcription assays using C. crescentus
E
32 RNA polymerase holoenzyme and in vivo studies using
rpoH-lacZ transcription fusions have confirmed the identity
of P2 as a
32-dependent promoter (30). The
levels of
32 increase transiently during heat shock in
C. crescentus, and the increased transcription of
rpoH seems to account for the induction of
32
levels (21, 30). This mode of regulation for C. crescentus
32 differs from that of its E. coli counterpart, whose complex regulatory region does not include
a
32-dependent promoter (7).
The C. crescentus groESL operon has been characterized and
shown to be subject to a dual type of control (2). Besides
being heat shock inducible, its expression is cell cycle regulated
during growth at normal temperatures. The results of primer extension analysis suggested the presence of two putative promoters regulating the expression of groESL, with only one (P2) presenting
characteristics of a
32-transcribed promoter and being
induced by heat shock. Furthermore, a CIRCE element was found
downstream of P2, suggesting that both a
32 promoter and
the IR element regulate the expression of groESL in
Caulobacter (2).
In this report, we confirm the
32-dependent expression
of groESL by overexpressing the C. crescentus
heat shock sigma factor using a multicopy plasmid and showing that the
increase in
32 levels results in an increase in the
amount of GroEL and an increase in the amount of the transcript coming
from the
32-like promoter. Furthermore, the role of the
CIRCE element in the regulation of the Caulobacter groESL
operon was investigated by using site-directed mutagenesis to obtain
mutations in this cis-acting element. The groE
regulatory regions containing each of these mutations were fused to a
promoterless lacZ gene, and expression of
-galactosidase
was analyzed in C. crescentus cells harboring these
transcription fusions after heat shock and throughout the cell cycle at
normal temperatures. A C. crescentus strain with a
disruption in the hrcA gene (22) was also
investigated for GroEL expression. Data obtained indicated that HrcA
and the CIRCE element are involved in cell cycle control of the
groESL operon.
| |
MATERIALS AND METHODS |
|---|
|
|
|---|
Bacterial strains and plasmids.
The synchronizable C. crescentus NA1000 (8) and LS2293 (hrcA)
(22) strains were grown in PYE (5) medium or in
modified minimal M2 glucose medium (15). Cultures were
synchronized by centrifugation through a Ludox gradient using a
modified version (6) of the procedure of Evinger and Agabian
(8). E. coli TG-1 was used for phage propagation
and cloning, and S17-1 was used as the donor strain in conjugations
with C. crescentus. Plasmid placZ/290
(10) contains a promoterless lacZ gene and was
used for transcriptional fusions with the C. crescentus
groESL regulatory region. Plasmids pTrc-His B (Invitrogen) and
pPROEX-1 (Gibco-BRL) were used for overexpressing C. crescentus GroEL and DnaK proteins, respectively, in E. coli. Plasmid pAR33 (21) is a derivative of the
multicopy plasmid pBBR1 (1) carrying the C. crescentus rpoH gene coding for
32 and its promoter region.
Plasmid pCS225 contains a lacZ transcription fusion with the
promoter of the xylX gene (17), and cells
carrying this plasmid were grown in PYE medium containing 0.1% xylose.
Site-directed mutagenesis of the groESL regulatory
regions and construction of lacZ transcription
fusions.
Site-directed mutagenesis was performed by the method of
Kunkel et al. (16), using a DNA fragment containing the
regulatory region of the groESL operon (2) cloned
in M13mp19 as the template. The synthetic oligonucleotides ML3
(5'-CCAGCGTCGTTGTTTCTCGAATGGAGC-3'), LRC
(5'-GAATGGAGCGACAAATAACAGTCCCGC-3'), LDR
(5'-GAATGGAGCGACAACAGTCCCGC-3'), MH10
(5'-CGAGGGCCGGCGGGGGACTCACCAGCG-3'), GP135
(5'-GAAGCGCTCCCCGCGAGCGCCCGAAAA-3'), and GP136
(5'-GAAGCTCCCCGCGCCGCGCCCGAA-3') were used to
obtain the site-directed mutations. The underlined bases correspond to the changes introduced in the wild-type sequence. The DNA fragments containing the mutations introduced by using ML3, LRC, LDR, MH10, GP135, and GP136 were then ligated to placZ/290, giving rise
to transcription fusions pRB19, pRB20, pRB22, pRB23, pRB35, and pRB36, respectively (for the location of each mutation, see Fig. 3). Transcription fusion pRB21 was obtained by doing a second round of
site-directed mutagenesis, using construct pRB19 as the template and
the synthetic oligonucleotide LRC. Plasmids containing these transcription fusions were introduced into C. crescentus by
conjugation with an E. coli donor (S17-1).
-Galactosidase
activity in mid-log-phase cultures was measured by the method of Miller
(18).
Western blots. Aliquots of C. crescentus cultures at 30°C or after heat shock at 42°C for different lengths of time were collected, and total protein was extracted as previously described (2). The protein extracts were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose membranes by the method of Towbin et al. (27). Analysis of the membranes was performed as previously described (2), except for the use of specific polyclonal antisera anti-GroEL or anti-DnaK and an enhanced chemiluminescence kit (Amersham). The anti-GroEL antiserum was obtained from a rabbit immunized with a fusion protein overexpressed in E. coli, corresponding to about 36 kDa of the carboxy-terminal portion of C. crescentus GroEL containing an N-terminal histidine tag from the expression vector pTrc-His B (Invitrogen). The anti-DnaK antiserum was obtained essentially the same way, but using expression vector pPROEX-1 (Gibco-BRL) and a DNA fragment encoding 65 kDa of the carboxy-terminal portion of C. crescentus DnaK.
Immunoprecipitation.
The temporal control of
groE-lacZ transcription fusions and GroEL synthesis was
investigated in synchronized cultures by pulse-labelling aliquots of
cells at different times of the cycle with
[35S]methionine for 5 min and immunoprecipitation of the
labelled proteins using either anti-
-galactosidase (Sigma) or
anti-GroEL antiserum, as previously described (12). As a
control of synchronization, antisera against C. crescentus
flagellins were also used. Similarly, the time course of heat shock
induction of
-galactosidase synthesis in groE-lacZ
transcription fusions was investigated by pulse-labelling mid-log-phase
cultures with [35S]methionine for 2 min and
immunoprecipitation of the labelled proteins using
anti-
-galactosidase antiserum.
Primer extension assay.
Mapping of the transcriptional start
sites was carried out by a primer extension assay. A synthetic
oligonucleotide (18-mer) complementary to nucleotides
15 to +3 (see
Fig. 2B) was 5' end labelled with [
-32P]ATP and
polynucleotide kinase and hybridized to 50 µg of total RNA isolated
from C. crescentus cells grown at 30°C or after a 15-min
exposure to 42°C. Annealing was carried out at 50°C for at least
4 h in 25 µl of 100 mM
piperazine-N,N'-bis(2-ethanesulfonic acid)
(PIPES) buffer (pH 7.0) containing 1 M NaCl and 5 mM EDTA. The nucleic
acids were ethanol precipitated and resuspended in 50 µl of 25 mM
Tris-HCl (pH 8.3) containing 3 mM MgCl2, 75 mM KCl, 1 mM
dithiothreitol, 40 U of RNase inhibitor, and 1 mM (each) dATP, dCTP,
dGTP, and dTTP. The annealed primer was extended at 37°C for 90 min
using 300 U of Moloney murine leukemia virus reverse transcriptase
(Amersham). RNA was digested for 30 min at 37°C by the addition of 1 µg of RNase A, and the extended products were analyzed by
electrophoresis on denaturing sequencing gels followed by
autoradiography.
| |
RESULTS |
|---|
|
|
|---|
C. crescentus groESL operon expression is controlled by
32.
To demonstrate that the C. crescentus
32 homolog regulates expression of the
groESL operon, we analyzed a C. crescentus strain carrying a high-copy-number plasmid (pAR33) containing the
32 gene and its promoter region (21). For
cells grown at 30°C, the levels of
32 in NA1000 cells
harboring pAR33 were investigated by Western blotting using antiserum
against the E. coli
32 and shown to be about
eightfold higher than those in NA1000 cells lacking the plasmid (Fig.
1). After heat shock,
32
levels were twofold higher in the strain carrying pAR33 than in the
control strain (Fig. 1). Since in vitro transcription studies have
shown that the purified C. crescentus
32
protein in the presence of RNA polymerase core enzyme specifically recognizes the heat shock-regulated promoter P1 of the C. crescentus dnaK gene (29), we investigated the levels
of DnaK protein in the
32-overexpressing strain. As
shown in Fig. 1, the amount of DnaK in this strain was about fivefold
higher in cells growing at 30°C than in the wild-type strain. During
the first 30 min after heat shock at 42°C, DnaK protein levels were
also higher in the
32-overexpressing strain (about
twofold). After that, the amount of DnaK became equal to or lower than
that in the control strain subjected to heat shock, probably reflecting
the fact that the levels of
32 decrease faster in the
overexpressing strain under these conditions (Fig. 1).
|
32-overexpressing strain, and as shown in Fig. 1, for
cultures grown at 30°C, GroEL levels were sixfold higher in this
strain than in the control. After heat shock, GroEL levels were higher
(two- to fivefold) in the strain harboring pAR33 up to 45 min of
exposure to 42°C. For longer periods of heat shock, as observed for
DnaK, the amount of GroEL was determined to be lower in the
32-overexpressing strain.
To investigate if the increase in the amount of GroEL was due to
increased transcription of the groESL operon, primer
extension experiments were performed by using an 18-nucleotide
synthetic oligomer complementary to nucleotides
15 to +3 (Fig.
2) and RNA was isolated from cells of
NA1000 strain carrying or not carrying the multicopy plasmid pAR33,
grown at 30°C or after 15 min at 42°C, as described in Materials
and Methods. As shown in Fig. 2, the amounts of transcript initiating
at the
32-like promoter were about 10-fold higher in
32-overexpressing cells growing at 30°C and about
7-fold higher when the cells were subjected to a 15-min heat shock at
42°C compared to cells with normal levels of
32, as
determined by densitometry scanning of the autoradiogram (not shown).
|
Analysis of transcriptional fusions containing mutations in the
CIRCE element.
In order to investigate the role of the CIRCE
element in expression of the C. crescentus groESL operon,
mutations in either one or both arms of the inverted repeat were
constructed by site-directed mutagenesis and the mutated
groESL regulatory regions were subcloned into the
transcription reporter vector placZ/290. As can be observed in Fig. 3, when three bases were changed
on either the left arm or the right arm of the inverted repeat, as in
transcription fusions pRB19 and pRB20, higher levels of
-galactosidase activity were observed for cells grown at 30°C.
These levels were about 50% higher than those observed in the
wild-type construct pMA11. In both cases, however, the levels of
-galactosidase activity still increased after exposure of the cells
to 40°C for 1 h. When both arms were mutated, reconstructing the
dyad symmetry of the inverted repeat but with a different nucleotide
sequence, as in pRB21, the levels of
-galactosidase activity were
even higher at 30°C, reaching 240% of the value obtained for the
wild-type construct pMA11, and here also
-galactosidase levels
increased still further after exposure of the cells to 40°C, reaching
levels 30% higher than those observed after heat shock with the
wild-type transcription fusion pMA11. Similar results were obtained
when four bases were deleted in the right arm of the inverted repeat,
as in transcription fusion pRB22. These results indicate that it is not
the formation of a putative stem-loop structure that regulates
expression of groESL, since in all four mutants, such a
structure could still be formed, as determined by using the FOLDRNA
program of the Genetics Computer Group package (not shown). Instead,
the data suggest that a protein or proteins probably bind to the
inverted repeat in a sequence-specific manner, negatively regulating
expression of groESL at normal temperatures.
|
10 region
of the heat shock promoter P2 lead to very low levels of
-galactosidase activity, similar to the values obtained with the
vector alone. Mutations in the
35 region of the P1 promoter, however,
do not result in lower levels of transcriptional activity, since the
values of
-galactosidase activity determined for pRB35 and pRB36
were found to be higher than that of the wild-type construct either at
30°C or after 1 h at 40°C. These results seem to indicate that
if P1 is a real promoter, it does not contribute significantly to
transcription of the groESL operon, either at 30°C or
during heat shock. For a control, we used the xylX-lacZ
transcription fusion where the promoter region of the xylX
gene, which is required for growth on xylose and is not heat shock
inducible, was subcloned into placZ/290, giving rise to
plasmid pCS225 (17). We observed that
-galactosidase
activity in this case does not increase after heat shock (Fig. 3).
To confirm that the promoter constructs containing mutations in the
CIRCE element are still heat shock inducible, the rate of synthesis of
-galactosidase was determined in cells harboring pMA11, pRB19, or
pRB22, after different lengths of exposure to 40°C. As shown in Fig.
4, the rate of
-galactosidase
synthesis increases significantly after heat shock in cells harboring
each of the three constructs. In the case of pRB22, for instance, the pattern of heat shock induction of
-galactosidase synthesis is similar to that observed for the wild-type construct pMA11. In cells
harboring pRB19, the rate of
-galactosidase synthesis presents a
lower but significant level of induction (3.5-fold) after heat shock.
The fact that the amount of induction observed with pRB19 does not
reach wild-type levels could be due to a decrease in the half-life of
the mRNA produced during heat shock, which is suggested by an
apparently faster turnoff of the response in this case.
|
Role of the CIRCE element in cell cycle control of
groESL expression.
We had previously shown that
sequences upstream of the heat shock promoter (P2) were not necessary
for cell cycle control of the groESL operon (2).
Since the
32 protein was shown to be synthesized
constitutively throughout the cell cycle at 30°C (21), we
decided to investigate the role of the CIRCE element in
groESL temporal control. Synthesis of
-galactosidase
directed by the groESL regulatory region carrying mutations
in the CIRCE element was analyzed throughout the C. crescentus cell cycle. To measure the rate of
-galactosidase synthesis, samples of synchronized NA1000
cultures harboring the groESL-lacZ transcription fusion
pMA11, pRB19, pRB20, or pRB21 were pulse-labelled with
[35S]methionine at different times during the cell cycle,
and extracts of these cells were immunoprecipitated with
anti-
-galactosidase antibody.
-galactosidase synthesis, which is temporally controlled in the
wild-type construct (pMA11), becomes constant throughout the cell cycle
when the CIRCE element carries a mutation in the left arm (pRB19), the
right arm (pRB20), or in both arms of the inverted repeat (pRB21). As
an internal control for cell synchrony, the rate of synthesis of the
flagellins was determined in all experiments. In Fig. 5, we present the
experiment done with cells harboring the pRB21 construct, showing the
normal pattern of flagellin synthesis. These results indicate that
groESL-lacZ transcription fusions carrying mutations in the
CIRCE element do not present cell cycle control of
-galactosidase
synthesis.
|
Disruption of the C. crescentus hrcA gene leads to constitutive expression of groESL at physiological temperatures. The C. crescentus hrcA gene encodes a protein with homologs reported for several other bacteria (22, 25) and was shown both in Caulobacter and in B. subtilis to be involved in the regulation of heat shock operons containing the CIRCE element (22, 31, 33). We have previously shown that in C. crescentus disruption of the hrcA gene increased transcription of the groESL operon ninefold at physiological temperatures with no significant effect on transcription after heat shock (22). This effect on transcription was not observed in the dnaKJ operon of Caulobacter, which does not present a CIRCE element in its regulatory region (2, 11, 22). Higher levels of groESL mRNA observed in cells of the hrcA mutant strain growing at normal temperatures are accompanied by an increase in the levels of GroEL protein, which were observed in Western blots using anti-GroEL antibody. As shown in Fig. 6, hrcA null mutant cells growing at 30°C present increased levels of GroEL (about fourfold) compared to those of the wild-type strain at the same temperature. The amount of GroEL still increased after heat shock, reaching similar levels in both mutant and wild-type cells after 45 min at 42°C, compatible with the data on transcription. Consistent with the fact that no increase in dnaKJ transcription is observed in hrcA mutant cells growing at 30°C compared to wild-type cells (22), no increase in the levels of DnaK was observed when the same Western blot was analyzed with anti-DnaK antibody (Fig. 6).
|
|
|
-galactosidase synthesis in synchronized cultures of strain LS2293
harboring the wild-type transcription fusion pMA11. As shown in Fig.
9, the rate of
-galactosidase
synthesis is no longer cell cycle controlled in the hrcA
null strain, indicating that transcription directed by the
groESL regulatory region becomes constitutive in the absence
of HrcA protein.
|
Enhanced transcription in CIRCE and hrcA mutants is
from the
32 promoter.
In order to investigate from
which promoter the increased transcription directed by the
groESL regulatory region was occurring in the
hrcA null strain and in the CIRCE element mutants, primer extension experiments were carried out with an 18-nucleotide synthetic oligomer complementary to nucleotides
15 to +3 (Fig.
10C) and RNA isolated from cells of
different mutant strains grown at 30°C or subjected to heat shock at
42°C for 30 min, as described in Materials and Methods. As shown in
Fig. 10, a single transcription start site was observed in all mutant
strains, which coincides with the initiation site highly induced during
heat shock, indicating that in all cases enhanced transcription was
occurring from the
32 promoter (P2). The fact that no
transcript originating from the putative P1 promoter was observed in
these experiments, in conjunction with the data using site-directed
mutagenesis (Fig. 3), strongly suggests that P1 might not be a true
promoter.
|
| |
DISCUSSION |
|---|
|
|
|---|
The initial description of a highly conserved inverted repeat
sequence, the CIRCE element, in front of the dnaKJ and
groESL operons of the gram-positive bacteria B. subtilis and C. acetobutylicum, in association with a
vegetative
70 promoter, led to the idea that this
inverted repeat was substituting for a
32 promoter and
therefore would be found only when
32 was absent.
Nevertheless, it soon became clear that the CIRCE element is quite
widespread, being found in the dnaK and/or groE operons of numerous phylogenetically distant bacteria (24), including bacterial species containing
32-mediated heat
shock regulation (2, 20, 25). In particular, this report
presents evidence of an operon controlled by a
32
promoter and the CIRCE element combined: the C. crescentus
groESL operon.
Previous characterization of the groESL regulatory region
had indicated the existence of two putative promoters, P1 and P2, corresponding to two transcriptional start sites, determined in primer
extension experiments (2). Only P2 was highly induced during
heat shock and presented
10 and
35 sequences conforming to the
consensus sequence of
32 promoters. In the present
study, we show that mutations in the putative
35 region of P1 do not
affect expression of the groESL-lacZ transcription fusion,
either in cells growing at 30°C or during heat shock. Furthermore,
primer extension experiments under conditions somewhat different from
those previously used (2) did not confirm the transcription
initiation site corresponding to the P1 promoter. Together these
results indicate that P1 might not be a real promoter. We also showed
that a mutation in the putative
10 region of the P2 promoter gave
rise to very low levels of
-galactosidase activity, which were close
to the levels determined for the reporter vector alone, indicating that
the heat shock-inducible promoter P2 is essential for transcriptional
activity, at physiological temperatures and during heat shock.
Furthermore, the observation that cells harboring extra copies of the
C. crescentus
32 gene presented a significant
increase in GroEL levels (sixfold at 30°C), similar to that observed
for the DnaK protein whose gene has been shown by in vitro
transcription studies to be
32 dependent
(29), and the results of primer extension experiments showing that the amount of the transcript initiating at the
32-like promoter is increased in the
32-overexpressing strain corroborate the hypothesis that
P2 is a
32 promoter.
Besides the
32-like promoter, the groESL
operon has a highly conserved CIRCE element located downstream of P2
(2). In C. crescentus, only the groESL
operon, not the dnaKJ operon, contains the CIRCE element,
(2, 3). A third heat shock operon was recently isolated in
C. crescentus (22); this operon encodes the
protein GrpE, which works in conjunction with DnaK, DnaJ, and HrcA,
which is the putative repressor that binds to the CIRCE element. The
hrcA-grpE operon regulatory region contains a
32 promoter but no CIRCE element (22).
Consistent with the presence of the CIRCE element in the regulatory region of the groESL operon, its expression is altered in a hrcA null mutant. Higher groESL mRNA levels were observed in hrcA mutant cells growing at physiological temperatures than in hrcA+ cells (22). As expected, no differences were detected in dnaKJ mRNA levels in hrcA and hrcA+ cells. In addition, no significant differences were observed in mRNA levels of both operons during heat shock when hrcA+ and hrcA cells were compared, suggesting a role for hrcA only at normal growth temperatures.
In the present report, we complemented these studies by looking at expression at the protein level. In agreement with a negative regulatory role for hrcA in CIRCE-containing operons, GroEL protein levels were observed to be fourfold higher in hrcA mutant cells than in hrcA+ cells growing at physiological temperatures, whereas no significant differences were observed in DnaK protein levels in both strains. GroEL levels increased further after heat shock, reaching similar levels in hrcA+ and hrcA cells after 30 min of exposure to 42°C. In addition, we showed that the rate of GroEL synthesis increases during heat shock to the same extent in the hrcA null mutant (threefold) and in wild-type cells (fourfold). These results are compatible with a repressor role for HrcA in cells growing at physiological temperatures.
Furthermore, we show here that point mutations in the CIRCE element located in the left arm, the right arm, or both arms of the inverted repeat, as well as a four-base deletion in the right arm, give rise to higher levels of expression of groESL-lacZ transcription fusions (50 to 240% higher) in cells growing at 30°C, with no significant effect on expression in cells subjected to heat shock. The data presented also indicated that HrcA binds to the CIRCE element in a sequence-specific manner.
The experiments with the hrcA null mutant and the
site-directed mutations in the inverted repeat suggested a role for
CIRCE and HrcA under non-heat shock conditions, and indeed the cell cycle-regulated expression groESL observed at physiological
temperatures (2) is lost when either the CIRCE element is
mutated or when the hrcA gene product is not present.
Moreover, enhanced transcription observed at physiological temperatures
in hrcA and CIRCE mutants was shown to occur from the
32-like promoter.
How the CIRCE element and the product of the hrcA gene control temporal expression of the groESL operon remains to be determined. The rate of C. crescentus hrcA transcription, determined by using a hrcA-lacZ transcription fusion, did not show significant variations during the cell cycle (22). The levels of HrcA protein, however, have not been determined. A possible cell cycle-dependent posttranslational activation or inactivation of HrcA should also be investigated. For instance, the cell cycle-regulated expression of Caulobacter late flagellar genes is due to a temporally controlled posttranslational activation of the transcriptional regulator FlbD (28). A similar type of control could be occurring with HrcA.
The reason why groESL expression is under cell cycle control in C. crescentus is not known. However, the roles of GroEL and GroES in the assembly of multiprotein complexes and the increase in GroEL levels observed in predivisional cells (2), coincident with the time of flagellum biogenesis, suggest a possible explanation for cell cycle control of groESL expression. The description of an E. coli groEL mutant with a defect in motility strengthens this hypothesis (4). A similar study with the characterization of C. crescentus groEL mutants is presently being undertaken to investigate a possible role for GroEL in flagellum assembly.
| |
ACKNOWLEDGMENTS |
|---|
This investigation was supported by grants to S.L.G. from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-PADCT). R.L.B. is a FAPESP predoctoral fellow, and M.A. is a CNPq predoctoral fellow. S.L.G. was partially supported by CNPq.
We thank L. Shapiro for the antiflagellin antiserum, A. C. Reisenauer for plasmid pAR33, R. C. Roberts for C. crescentus LS2293, U. Jenal for plasmid pCS225, M. V. Marques for critical reading of the manuscript, and Elisety de Andrade Silva for manuscript preparation.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP.26.077, São Paulo, SP 05599-970, Brasil. Phone: 55-11-815-3286. Fax: 55-11-818-7986. E-mail: sulgomes{at}quim.iq.usp.br.
| |
REFERENCES |
|---|
|
|
|---|
| 1. | Antoine, R., and C. Locht. 1992. Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from gram-positive organisms. Mol. Microbiol. 6:1785-1799[Medline]. |
| 2. | Avedissian, M., and S. L. Gomes. 1996. Expression of the groESL operon is cell cycle controlled in Caulobacter crescentus. Mol. Microbiol. 19:79-89[Medline]. |
| 3. |
Avedissian, M.,
D. Lessing,
J. W. Gober,
L. Shapiro, and S. L. Gomes.
1995.
Regulation of the Caulobacter crescentus dnaKJ operon.
J. Bacteriol.
177:3479-3484 |
| 4. |
Burnett, B. P.,
A. L. Horwich, and K. B. Low.
1994.
A carboxy-terminal deletion impairs the assembly of GroEL and confers a pleiotropic phenotype in Escherichia coli K-12.
J. Bacteriol.
176:6980-6985 |
| 5. | Contreras, I., L. Shapiro, and S. Henry. 1987. Membrane phospholipid composition of Caulobacter crescentus. J. Bacteriol. 135:1130-1136. |
| 6. |
Dingwall, A.,
J. W. Gober, and L. Shapiro.
1990.
Identification of a Caulobacter basal body structural gene and a cis-acting site required for activation of transcription.
J. Bacteriol.
172:6066-6076 |
| 7. |
Erickson, J. W.,
V. Vaughn,
W. A. Walter,
F. C. Neidhardt, and C. A. Gross.
1987.
Regulation of the promoters and transcripts of rpoH, the Escherichia coli heat shock regulatory gene.
Genes Dev.
1:419-432 |
| 8. |
Evinger, M., and N. Agabian.
1977.
Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells.
J. Bacteriol.
132:294-301 |
| 9. | Gamer, J., G. Multhaup, T. Tomoyasu, J. S. McCarty, S. Rudiger, H. J. Schonfeld, C. Schirra, H. Bujard, and B. Bukau. 1996. A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulate activity of the Escherichia coli heat shock transcription factor sigma 32. EMBO J. 15:607-617[Medline]. |
| 10. | Gober, J. W., and L. Shapiro. 1992. A developmentally regulated Caulobacter flagellar promoter is activated by 3' enhancer and IHF binding elements. Mol. Biol. Cell 3:913-916[Abstract]. |
| 11. |
Gomes, S. L.,
J. W. Gober, and L. Shapiro.
1990.
Expression of the Caulobacter heat shock gene dnaK is developmentally controlled during growth at normal temperatures.
J. Bacteriol.
172:3051-3059 |
| 12. | Gomes, S. L., and L. Shapiro. 1984. Differential expression and position of chemotaxis methylation proteins in Caulobacter. J. Mol. Biol. 77:551-568. |
| 13. | Hecker, M., W. Schumann, and V. Völker. 1996. Heat-shock and general stress response in Bacillus subtilis. Mol. Microbiol. 19:417-428[Medline]. |
| 14. |
Herman, C.,
D. Thévenet,
R. D'Ari, and P. Bouloc.
1995.
Degradation of 32, the heat shock regulator in Escherichia coli, is governed by HflB.
Proc. Natl. Acad. Sci. USA
92:3516-3520 |
| 15. |
Johnson, R. C., and B. Ely.
1977.
Isolation of spontaneously derived mutants of Caulobacter crescentus.
Genetics
86:25-32 |
| 16. | Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154:367-382[Medline]. |
| 17. |
Meisenzahl, A. C.,
L. Shapiro, and U. Jenal.
1997.
Isolation and characterization of a xylose-dependent promoter from Caulobacter crescentus.
J. Bacteriol.
179:592-600 |
| 18. | Miller, J. H. 1972. . Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 19. |
Narberhaus, F., and H. Bahl.
1992.
Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum.
J. Bacteriol.
174:3282-3289 |
| 20. |
Narberhaus, F.,
W. Weiglhofer,
H.-M. Fischer, and H. Hennecke.
1996.
The Bradyrhizobium japonicum rpoH1 gene encoding a 32-like protein is part of a unique heat shock gene cluster together with groESL1 and three small heat shock genes.
J. Bacteriol.
178:5337-5346 |
| 21. |
Reisenauer, A. C.,
C. D. Mohr, and L. Shapiro.
1996.
Regulation of a heat shock 32 homolog in Caulobacter crescentus.
J. Bacteriol.
178:1919-1927 |
| 22. |
Roberts, R. C.,
C. Toochinda,
M. Avedissian,
R. L. Baldini,
S. L. Gomes, and L. Shapiro.
1996.
Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE.
J. Bacteriol.
178:1829-1841 |
| 23. |
Schulz, A., and W. Schumann.
1996.
hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes.
J. Bacteriol.
178:1088-1093 |
| 24. |
Segal, G., and E. Z. Ron.
1996.
Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat.
J. Bacteriol.
178:3634-3640 |
| 25. | Segal, G., and E. Z. Ron. 1996. Regulation and organization of the groE and dnaK operons in Eubacteria. FEMS Microbiol. Lett. 138:1-10[Medline]. |
| 26. |
Tomoyasu, T.,
J. Gamer,
B. Bukau,
M. Kanemori,
H. Mori,
A. J. Rutman,
A. B. Oppenheim,
T. Yura,
K. Yamanaka,
H. Niki,
S. Hiraga, and T. Ogura.
1995.
Escherichia coli FtsH is a membrane-bound ATP-dependent protease which degrades the heat-shock transcription factor 32.
EMBO J.
14:2551-2560[Medline].
|
| 27. |
Towbin, H.,
T. Staehelin, and J. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:4350-4354 |
| 28. |
Wingrove, J. A.,
E. K. Mangan, and J. W. Gober.
1993.
Spatial and temporal phosphorylation of a transcriptional activator regulates pole-specific gene expression in Caulobacter.
Genes Dev.
7:1979-1992 |
| 29. |
Wu, J., and A. Newton.
1996.
Isolation, identification, and transcriptional specificity of the heat shock sigma factor 32 from Caulobacter crescentus.
J. Bacteriol.
178:2094-2101 |
| 30. |
Wu, J., and A. Newton.
1997.
The Caulobacter heat shock sigma factor gene rpoH is positively autoregulated from a 32-dependent promoter.
J. Bacteriol.
179:514-521 |
| 31. |
Yuan, G., and S.-L. Wong.
1995.
Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK.
J. Bacteriol.
177:6462-6468 |
| 32. | Yura, T., H. Nagai, and H. Mori. 1993. Regulation of the heat-shock response in Bacteria. Annu. Rev. Microbiol. 47:321-350[Medline]. |
| 33. |
Zuber, V., and W. Schumann.
1994.
CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis.
J. Bacteriol.
176:1359-1363 |
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||