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Journal of Bacteriology, March 1999, p. 1603-1609, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
CspI, the Ninth Member of the CspA Family of
Escherichia coli, Is Induced upon Cold Shock
Nan
Wang,
Kunitoshi
Yamanaka, and
Masayori
Inouye*
Department of Biochemistry, Robert Wood
Johnson Medical School, University of Medicine and Dentistry of New
Jersey, Piscataway, New Jersey 08854
Received 17 September 1998/Accepted 10 December 1998
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ABSTRACT |
Escherichia coli contains the CspA family, consisting
of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be
stationary-phase inducible. The cspI gene is located
at 35.2 min on the E. coli chromosome map, and CspI shows
70, 70, and 79% identity to CspA, CspB, and CspG, respectively.
Analyses of cspI-lacZ fusion constructs and the
cspI mRNA revealed that cspI is cold shock
inducible. The 5'-untranslated region of the cspI mRNA
consists of 145 bases and causes a negative effect on cspI
expression at 37°C. The cspI mRNA was very unstable at
37°C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is
maximal at or below 15°C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for
their induction are different: CspA induction occurs over the broadest
temperature range (30 to 10°C), CspI induction occurs over the
narrowest and lowest temperature range (15 to 10°C), and CspB and
CspG occurs at temperatures between the above extremes (20 to 10°C).
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INTRODUCTION |
When Escherichia coli
cells grown at 37°C are transferred to low temperatures such as
15°C, a set of proteins called cold shock proteins are transiently
induced at very high levels during a growth lag period called the
acclimation phase (16, 17, 30). Among them, CspA has been
identified as a major cold shock protein, consisting of 70 amino acid
residues. It has two RNA binding motifs, RNP1 and RNP2 (10,
16), and forms a 
-barrel structure (7, 24, 27).
CspA was revealed to cooperatively bind to RNAs and single-stranded
DNAs and is considered to function as an RNA chaperone, which may
prevent the formation of secondary structures of mRNAs for efficient
translation at low temperatures (15).
The cspA promoter is highly active at 37°C, although CspA
protein is hardly detected at this temperature (5, 22),
indicating that cspA expression at low temperatures is
regulated posttranscriptionally. It should be mentioned, however, that
the AT-rich sequence immediately upstream of the 
35 region of the
cspA promoter functions as a UP element to enhance
cspA transcription (9, 22). The cspA mRNA is extremely unstable at 37°C but is dramatically stabilized upon cold shock (3, 5, 8, 22). The cspA mRNA
possesses an unusually long 5' untranslated region (5'-UTR) consisting
of 159 bases (29). Mutation analyses have shown that the
5'-UTR of the cspA mRNA plays a crucial role in its cold
shock inducibility (5, 22). In addition, translation
initiation of the cspA mRNA appears to be very efficient at
low temperature in comparison with mRNAs of non-cold-shock proteins
(22). Thus, the cspA expression is regulated in a
complex manner, that is, at the levels of transcription, mRNA stability
and translation efficiency (3, 5, 8, 9, 22).
cspA induction occurs transiently during the acclimation
phase upon cold shock, as mentioned above. At the end of the
acclimation phase, cspA expression is repressed to a new
basal level (10). It has been proposed that the cold box
sequence, which is located in the 5'-UTR, and a factor which might bind
to the cold box to repress the cspA expression at the level
of transcription are responsible for autoregulation of cspA
expression (6, 14).
Six additional cspA homologues in E. coli,
cspB to cspG, have been identified by means of
Southern analysis with cspA as a probe (cspB and
cspC [19]), isolation of multicopy
suppressors of a chromosome partition mutant (cspC and
cspE [35]) and cold shock induction
(cspG [23]). Of these, cspA,
cspB, and cspG are cold shock inducible (10,
19, 23) and cspC and cspE are expressed at
both high and low temperatures (35). cspD is located upstream of clpA (11) and is induced
during stationary phase and upon nutrition starvation (34).
cspF is closely linked to cspB (33);
however, its function is not known. cspA, cspB, and cspG mRNAs share a highly conserved, long 5'-UTR
sequence (19, 23, 29), suggesting that the expression of
cspB and cspG is regulated in a similar way to
that of cspA. It should be noted, however, that CspB and
CspG are induced in a narrower range of low temperature than is CspA
(4). cspA is dispensable for growth at either 37 or 15°C, and the production of CspB and CspG increased in a
cspA deletion strain, suggesting that they may have similar
functions (2).
Upon completion of E. coli genome sequencing, two more CspA
homologues, designated CspH and CspI (33), were found. They show the highest similarity to CspF and CspG, respectively
(33). Although the primary amino acid sequence of CspI shows
high identity not only to CspG (79%) but also to CspA (70%) and CspB
(70%) (33), the region corresponding to the highly
conserved 5'-UTR of cspA, cspB, and
cspG is less highly conserved in cspI.
Here we demonstrate that cspI is another cold shock gene, as
judged by lacZ expression of both the transcriptional and
the translational cspI-lacZ fusions and by CspI production,
determined by two-dimensional gel electrophoresis. As shown for
cspA, the cspI mRNA was dramatically stabilized
upon cold shock and the overproduction of the cspI 5'-UTR
caused derepression of cspA, cspB,
cspG, and cspI. In addition, the cspI
gene was induced in the lowest temperature range (15 to 10°C),
suggesting that CspI may play an important physiological role in growth
at very low temperature.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
E. coli JM83
(32) and AR134 (MC4100 pcnB80) (13)
were used. Plasmid pUC19 (32) was used for cloning. Plasmids
pRS414 and pRS415 (28) were used for construction of
lacZ fusions. Plasmid pCspA-LacZ, in which the
cspA upstream region and the first 13 codons of
cspA were fused translationally to lacZ on the
pRS414 vector, as described previously (19), was used as a control.
Luria-Bertani (LB) and M9 media, supplemented with 0.4% glucose, 0.4%
Casamino Acids, and 50 µg/ml of thiamine, were used for bacterial
growth. When necessary, ampicillin was added at a final concentration
of 50 µg/ml.
General techniques.
DNA cloning was carried out by the
method described previously (26). PCR amplification was
carried out as specified in the manufacturer's instruction manual
(Boehringer) with 30 cycles of amplification steps each of 1 min at
95°C, 2 min at 50°C, and 2 min at 72°C. Restriction enzymes and
DNA modification enzymes were purchased from Boehringer, Gibco BRL, and
New England Biolabs.
Plasmid construction.
cspI was amplified by PCR with
synthetic oligonucleotide primers, 8188 (5'-aagaattcAACATTTACATCGCGGAA-3') and 8187 (5'-ttgggatCCTCAAAGCGCCACTA-3'), where the 5'
tails are shown in lowercase type and the EcoRI and BamHI sites, respectively, are underlined. Genomic DNA from
prototype strain W3110 (18) was used as a template. The PCR
fragment was directly cloned into the SmaI site of pUC19,
yielding pNWI2. To construct a plasmid that can express only the 5'-UTR
of cspI but not its coding region, pNWI2 was digested with
StyI and SacI, blunt ended with T4 DNA
polymerase, and then self-ligated, yielding pNWI6.
To construct the transcriptional and translational
cspI-lacZ
fusions, PCR was first carried out with primers 8188 (see above)
and
8010 (5'-ggg
ggatccGGGTTAAACCATTTCACT-3'), where
the 5' tail
is shown in lowercase type and the
BamHI site is
underlined. The
PCR fragment was digested with
EcoRI and
BamHI and then cloned
into pRS414 for the construction of a
translational fusion and
into pRS415 for the construction of a
transcriptional fusion,
yielding pNWI3 and pNWI4, respectively. For the
deletion construct
of the entire 5' untranslated region of the
cspI-lacZ fusion,
PCR was carried out with primers 8188 (see
above) and 8404 (5'-gg
ggatcCAGAACACCATTAACGC-3'),
where the 5' tail is shown in lowercase type and the
BamHI site
is underlined. The PCR fragment was cloned into
pRS415 in a similar
way to that described above, yielding pNWI5. All
the constructs
were confirmed by DNA sequencing with Sequenase version
2.0
(Amersham).
Assay for -galactosidase activity.
The
cspI-lacZ fusion constructs were introduced into strain
AR134. Cells were grown in LB or M9 medium at 37°C to mid-log phase
and then transferred to 15°C. Portions of culture were taken immediately before the temperature downshift (0 h) and at 1, 2, 3, and
5 h after the temperature downshift. -Galactosidase activity was measured as described by Miller (21). The assay was done at least in duplicate at each time point.
Isolation of RNA and primer extension.
Strain JM83 was grown
in LB medium at 37°C to mid-log phase and then transferred to 15°C.
RNA was extracted from a 1.5-ml culture by the hot-phenol method
described previously (1). Primer 8272 (5'-CCAAAACCTTTTTCAGGG-3') for detection of cspI
and primer 4593 (5'-ACATAGTGTATTACCTTTAA-3') for detection
of cspA were labeled with [
-32P]ATP
(>5,000 Ci/mmol; DuPont-New England Nuclear) by using T4 polynucleotide kinase (Gibco BRL). Primer extension was carried out
with 5 µg of RNA at 42°C for 1 h in a final volume of 10 µl, which contained 50 mM Tris-HCl (pH 8.5), 8 mM MgCl2, 30 mM
KCl, 1 mM dithiothreitol, 0.4 pmol of 32P-labeled primer,
0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dTTP, 10 U of RNase
inhibitor (Boehringer Mannheim), and 6.25 U of avian myeloblastosis
virus reverse transcriptase (Boehringer Mannheim). Primer extension
products were analyzed on a 6% polyacrylamide gel under denaturing conditions.
For measurement of the stability of mRNA at 15°C, rifampin was added
1 h after the temperature downshift to 15°C at a final
concentration of 200 µg/ml to stop transcription. For measurement
of
the stability of mRNA at 37°C, the culture was shifted to 15°C
for
30 min to accumulate mRNAs, a 5-ml sample was taken and mixed
with 5 ml
of the medium (which was prewarmed at 60°C) in a glass
flask kept at
37°C, rifampin was simultaneously added to a final
concentration of
200 µg/ml, a 1.5-ml sample was taken at each
time point, and RNA
extraction and primer extension assays were
done as described above.
Primer extension products were quantitated
by using a phosphorimager
(Bio-Rad).
Protein-labeling experiment.
Cells were grown in M9 medium
supplemented with glucose, 19 amino acids (no methionine), and thiamine
at 37°C, and then transferred to an indicated temperature. Cells were
labeled with [35S]methionine (1092 Ci/mmol; Amersham) for
30 min at lower temperatures and then chased for 5 min by adding
nonradioactive methionine to a final concentration of 0.2 M. Cell
lysates were prepared and processed by two-dimensional gel
electrophoresis as described previously (31).
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RESULTS AND Discussion |
Sequence comparison among CspA, CspB, CspG, and CspI.
The
E. coli genome-sequencing project has revealed the ninth
member of the CspA family, which we designated CspI (33).
CspI shows the highest identity to CspG (79%) (Fig.
1A), and on the phylogenetic tree it
belongs to the same group as CspA, CspB, and CspG, all of which are
cold shock inducible as described previously (33). The
three-dimensional structure of CspA has been determined. It consists of
five antiparallel -strands forming a -barrel structure with
two -sheets (7, 24, 27). CspI contains well-conserved hydrophobic residues including V9, I21, V30, V32, and
V51 (Fig. 1A), which form a hydrophobic core in CspA (7, 24,
27). In addition, two RNA binding motifs, RNP1 and RNP2, are well
conserved in CspI (Fig. 1A). These facts suggest that CspI may form a
conformation similar to that of CspA and may also bind to RNA and
single-stranded DNA, as CspA does (15).
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FIG. 1.
Sequence comparison of four cold shock-inducible members
of the E. coli CspA family. (A) Amino acid sequence
alignments of CspI (database accession no. AE000252), CspG (AE000201),
CspB (AE000252), and CspA (AE000433). Residues identical to CspI are
shown as dots. The residues forming the hydrophobic core in the
-barrel structure are indicated by solid circles above the
sequences. The RNA binding motifs, RNP1 and RNP2, are boxed. Their
amino acid sequence homologies are shown on the right, with CspI set at
100%. (B) Sequence alignment of the promoter, 5'-UTR, and the first 13 codon nucleotides of cspI, cspG, cspB,
and cspA. Nucleotides identical to cspI are shown
as dots. To maximize the alignment, some gaps have been introduced;
these are indicated by dashes. The transcription start sites are in
bold letters and are marked as +1. The translation start codon ATGs are
also in bold letters and are underlined. The most homologous sequences
(UP element, -35 region, -10 region, cold box, upstream sequence,
Shine-Dalgarno [SD] sequence, and downstream box) are boxed and
labeled above the boxes.
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Cold shock-inducible expression of cspI.
The
cspI gene is located at 35.2 min on the E. coli
chromosome and transcribed counterclockwise. To determine the
regulation of cspI expression, the 387-bp DNA fragment
containing the 346-bp upstream sequence of the cspI coding
region and the region for the first 13 codons of cspI was
amplified and cloned, to translationally and transcriptionally fuse to
lacZ on pRS414 and pRS415, yielding pNWI3 and pNWI4,
respectively. These fusion constructs were introduced into AR134, and
the -galactosidase activity was measured. AR134 is a pcnB
mutant (13), which keeps pBR322 derivatives at low copy
number (20) to minimize any positive or negative effects of
the multicopy plasmids on cspI-lacZ expression.
-Galactosidase activities of both fusion constructs were very low at
37°C at mid-log phase (zero time point in Fig.
2). Since
CspI shows the highest identity
to cold shock-inducible CspG,
we analyzed the effect of temperature
downshift on
cspI-lacZ expression.
After the temperature
downshift from 37 to 15°C, the
-galactosidase
activities of both
the transcriptional and the translational fusion
constructs
dramatically increased and were both approximately
twofold higher than
that of the
cspA-lacZ translational fusion,
as shown in Fig.
2, indicating that
cspI is a cold shock-inducible
gene like
cspA,
cspB, and
cspG.
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FIG. 2.
Cold shock induction of -galactosidase activity.
Strain AR134, harboring various plasmids, was grown to mid-log phase at
37°C in LB medium containing ampicillin (50 µg/ml) and then
transferred to 15°C. Samples were taken at 0, 1, 2, 3, and 5 h
after the temperature downshift, and -galactosidase activity was
measured. The assay was carried out at least in duplicate at each time
point. Symbols:  , pNWI3;  , pNWI4;  , pCspA-LacZ;  , pRS414.
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Although the expression of
cspA,
cspB,
cspG, and
cspI is cold shock inducible and the
primary amino acid sequence of CspI
is highly homologous to those of
CspA, CspB, and CspG, a putative
5'-UTR sequence for
cspI
does not show high similarity to those
for
cspA,
cspB, and
cspG, suggesting that
cspI
might be regulated
somewhat differently. To identify the 5'-UTR of
cspI, the transcription
start site of
cspI was
determined by primer extension analysis
with primer 8272, which is
specific for
cspI. This primer corresponds
to the
complementary strand for
cspI codons 14 through 19. Total
RNA of strain JM83 was extracted from cells grown at 37°C and
from
cells grown for 0.5 and 3 h after the temperature downshift
to
15°C. Primer extension products of
cspI from
pre-cold-shocked
cells were hardly detected (Fig.
3, lane 2). However, at 0.5 h
after
the temperature downshift, the amounts of primer extension
products
dramatically increased (lane 3), indicating that the
amount of
cspI mRNA greatly increased upon cold shock. At 3 h
after the temperature downshift, the amount of
cspI mRNA was
reduced
to a new basal level (lane 4), which is slightly higher than
that
at 37°C. The pattern of cold shock induction of the
cspI mRNA
is very similar to those of the
cspA,
cspB, and
cspG mRNAs (
4,
23),
indicating that
cspI is also transiently induced upon cold
shock. The
cspI mRNA was not induced during stationary phase
at
37°C (lane 5).
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FIG. 3.
Primer extension analysis of the cspI mRNA.
Strain JM83 was grown in LB medium at 37°C. RNA extraction and primer
extension analysis were carried out as described in Materials and
Methods. The primer corresponds to the complementary strand for codons
14 to 19 of cspI. Lanes: 1, without RNA; 2, with RNA
extracted from exponentially growing cells at 37°C; 3, with RNA
extracted from cells at 0.5 h after the temperature downshift to
15°C; 4, with RNA extracted from cells at 3 h after the shift;
5, with RNA extracted from stationary-phase cells at 37°C. Primer
extension products were analyzed on a denatured polyacrylamide gel
together with a sequencing ladder. The sequence is shown at the right;
the arrow indicates the transcription start site.
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On the basis of the primer extension experiment described above, the
possible transcription start site and the deduced promoter
region, the
-35 and -10 sequences (TTGCTA and GTTAAT,
respectively)
are identified as shown in Fig.
1B. The promoter
sequence of
cspI is very similar to those of
cspA,
cspB, and
cspG. Upstream of
the
-35 region of
cspA,
cspB, and
cspG is
an AT-rich region called
the UP element (
25), which is
believed to play an important
role in maintaining the high promoter
activity of
cspA at both
37 and 15°C (
22). The
UP element also exists in the
cspI gene
and is likely to
play an important role in
cspI transcription
at low
temperature.
The primer extension experiment (Fig.
3) also reveals that the
cspI mRNA contains a long 5'-UTR consisting of 145 bases,
which
is comparable to the 5'-UTRs of
cspA (159 bases),
cspB (161 bases),
and
cspG (156 bases). Although
the sequence of the 5'-UTR of
cspI is not highly homologous
to those of
cspA,
cspB, and
cspG, the
5'-UTR of
cspI still contains a well-conserved motif, termed
the
cold box, which is believed to be involved in autoregulation at
the
end of the acclimation phase (
6,
14, see below).
Farther
downstream in the 5'-UTR, there is a 12-base conserved sequence
designated the upstream sequence, which may be involved in the
translation efficiency of
cspA (
36). It should be
noted that
cspA,
cspB, and
cspG all
have a downstream box downstream of the
translation initiation codon,
which has been shown to play an
important role in cold shock induction
at the level of translation
(
22). As shown in Fig.
1B,
cspI has exactly the same downstream
box as
cspB
and
cspG, suggesting that the
cspI downstream box
also plays an essential role in translation at low
temperature.
cspI mRNA stabilization upon cold shock.
Based on
the analysis of the cspI-lacZ fusion constructs,
cspI expression seems to be regulated by transcription
and/or mRNA stability (Fig. 2). It has been shown that mRNA stability
plays a critical role in the cold inducibility of cspA and
that the stability of mRNA is regulated by its long 5'-UTR (3, 5, 8). Therefore, we examined the stability of the cspI
mRNA at both 37 and 15°C by primer extension analyses. As shown in
Fig. 4, the cspI mRNA was very
unstable at 37°C, with a half-life of approximately 30 s,
somewhat more stable than the cspA mRNA (half-life, approximately 20 s). However, at 1 h after the temperature
downshift, the cspI mRNA and the cspA mRNA were
stabilized with half-lives of 14 and 12 min, respectively. These
results suggest that mRNA stability also plays a major role in the cold
shock induction of cspI.
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FIG. 4.
Analysis of mRNA stability. A culture of strain JM83
grown in LB medium at 37°C was shifted to 15°C. (A) For measurement
of mRNA stability at 37°C, a culture preincubated at 15°C for 30 min was shifted back to 37°C. Rifampin was added to the culture to a
final concentration of 200 µg/ml at the point of the temperature
upshift. RNAs were extracted at 0 (lanes 1 and 6), 1 (lanes 2 and 7), 2 (lanes 3 and 8), 3 (lanes 4 and 9), and 5 (lanes 5 and 10) min after
the addition of rifampin. (B) For measurement of the mRNA stability at
15°C, rifampin was added 1 h after the temperature downshift and
RNAs were extracted at 0 (lanes 1 and 6), 5 (lanes 2 and 7), 10 (lanes
3 and 8), 20 (lanes 4 and 9), and 30 (lanes 5 and 10) min after the
addition of rifampin. Primer extension was carried out with a primer
for cspI (lanes 1 to 5) and with a primer for
cspA (lanes 6 to 10) as described in Materials and Methods.
(C and D) Graphical presentations based on the results obtained in
panels A and B, respectively. The radioactivities of primer extension
products were measured and plotted, with the product at time zero set
to 100%. Symbols: , cspI; , cspA.
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When the 5'-UTR of the
cspA mRNA was deleted,
cspA expression was observed even at 37°C, indicating that
the 5'-UTR has a
negative effect on
cspA expression at
37°C (
22). To elucidate
the effect of
cspI
5'-UTR on
cspI expression, another transcriptional
cspI-lacZ fusion construct, pNWI5, which does not contain
the
5'-UTR, was prepared (see Materials and Methods). At 37°C, the
-galactosidase activity in the cells harboring pNWI5 was 980
U,
which was much higher than that in the cells harboring pNWI4
(29 U),
which contains the 5'-UTR. This indicates that the
cspI 5'-UTR has a negative effect on its own gene expression at 37°C,
as
the
cspA 5'-UTR
does.
CspI induction at lower temperatures.
CspA, CspB, CspG, and
CspI are all cold shock inducible. However, upon cold shock, the
temperature dependence of CspA induction is broader
while that of CspB and CspG is restricted to lower temperatures and to
a narrower temperature range (4). To examine the optimal
temperature for CspI induction, two-dimensional (2D) gel
electrophoresis was carried out with cells labeled with
[35S]methionine at different temperatures. For this
purpose, we first determined the spot corresponding to CspI by using a
plasmid which overproduces CspI. As shown in Fig.
5A, CspI migrated very close to CspB and
its production was indeed induced upon cold shock.
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FIG. 5.
Analysis of CspI protein by 2D gel electrophoresis. (A)
Cultures of strain JM83 harboring pUC19 or pNWI2 were labeled with
[35S]methionine at 37°C and labeled 30 min after the
temperature downshift to 15°C. Total-cell extracts were analyzed by
2D gel electrophoresis, and autoradiograms were obtained. Only a
portion corresponding to low-molecular-weight proteins is shown. (B)
Cultures of strain JM83 were labeled with [35S]methionine
30 min after the temperature downshift from 37°C to 30, 25, 20, 15, and 10°C, and the one indicated by "15°C 3 hr" is a sample
labeled at 3 h after the temperature downshift to 15°C. CspI is
indicated by an arrowhead. The positions of CspA, CspB, and CspG are
shown by labeled arrowheads in one of the panels in A and B: 1, CspA;
2, CspB; 3, CspG; and 4, CspI.
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CspI production from the chromosomal
cspI gene could not be
detected at 37°C but became clearly detectable at 15°C (Fig.
5B),
unambiguously demonstrating that
cspI is a cold
shock-inducible
gene. Next, cells grown at 37°C to mid-log phase was
transferred
to different temperatures, i.e., 30, 25, 20, 15, and
10°C. At
30 min after the temperature shift, cells were labeled with
[
35S]methionine for 30 min and total cellular proteins
were analyzed
by 2D gel electrophoresis. In contrast to CspA, CspB, and
CspG,
CspI was induced only when the temperature was shifted to or
below
15°C (Fig.
5B), indicating that CspI has the narrowest and
lowest
temperature range for its induction. CspI production was reduced
to a lower level at 3 h after the temperature downshift, as can
be
seen from the gel (Fig.
5B), which is in good agreement with
the
results of the primer extension analysis as shown in Fig.
3. This
indicates that CspI production is transiently induced
during the
acclimation phase upon cold shock, as for CspA, CspB,
and CspG (
4,
10,
23,
29).
It is interesting that the rate of CspI synthesis is much lower than
that of CspA (Fig.
5), although the
-galactosidase activity
of the
cspI-lacZ translational fusion construct is higher than
that
of the
cspA-lacZ translational fusion construct (Fig.
2).
In
both fusion constructs, the first 13 codons of
cspI or
cspA were translationally fused to
lacZ as
mentioned above. Since transcription
initiation and translation
initiation from the fusion constructs
are likely to occur in the same
manner as those from the chromosomal
copy of
cspA and
cspI, we examined any difference in protein stability
between CspA and CspI. Their half-lives were measured by pulse-chase
experiments 30 min after the temperature downshift and found to
be
longer than 5 h, although CspI was somewhat less stable than
CspA
(data not shown). The chasing in the 2D electrophoresis in
Fig.
5 was
only 5 min. Therefore, it is unlikely that the low
production of CspI
is due to protein stability. These results
therefore suggest that the
difference between CspA and CspI production
at low temperature may be
at the level of translation
elongation.
Derepression by overexpression of the cspA or
cspI 5'-UTR.
It has been reported that when the
cspA 5'-UTR was overproduced during cold shock treatment,
CspA and CspB expression were no longer transient and a high level of
CspA and CspB expression was still observed after the acclimation phase
(6, 14). This phenomenon, called derepression, was also
observed when the region from +1 to +25 of the cspA 5'-UTR
was overexpressed. Within this region, a highly
conserved sequence, designated the cold box sequence, was found (Fig.
1B) (14). Conversely, deletion of the cold box region
abolished the derepression effect of the cspA 5'-UTR
(6). It has been proposed that a factor might bind to the
cold box to repress the cspA expression at the level of
transcription at the end of the acclimation phase (6, 14).
The overproduction of the 5'-UTR containing the cold box is thus
expected to sequester this factor to derepress cspA expression.
As mentioned above, the
cspI 5'-UTR possesses a cold box
sequence. To investigate whether the
cspI cold box has a
similar
effect as that of
cspA, we constructed a plasmid
pNWI6 that is
able to overexpress the
cspI 5'-UTR from +1 to
+113. By primer
extension analysis, we confirmed that the
cspI 5'-UTR was effectively
overexpressed (data not shown).
pUC19-600 that can overexpress
the
cspA 5'-UTR
(
14) was used as a positive control, and pUC19
was used as a
negative control. Before and at 1 h after temperature
downshift,
the patterns of CspA, CspB, CspG, and CspI expression
were essentially
the same among cells harboring pUC19, pUC19-600,
and pNWI6 (Fig.
6A, panels a to f). At 3 h after the
temperature
downshift, all four proteins were greatly repressed
in cells harboring
pUC19 (Fig.
6A, panel g) while production of
all four proteins
became derepressed in cells overexpressing either the
cspA or
the
cspI 5'-UTR (Fig.
6A, panels h and i,
respectively). As shown
in Fig.
6B, overexpression of both the
cspA and the
cspI 5'-UTRs
has a derepression
effect, although the effect of the
cspI 5'-UTR
is weaker
than that of the
cspA 5'-UTR. These results suggest
that the
cspI 5'-UTR probably plays a role in the autoregulation
of
the
cspI gene at the end of the acclimation phase.
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|
FIG. 6.
Effect of cspI 5'-UTR overexpression on CspA,
CspB, CspG, and CspI expression. (A) Cultures of strain JM83 harboring
pUC19 (a, d, and g), pUC19-600 (b, e, and h), or pNWI6 (c, f, and i)
were labeled with [35S]methionine at 37°C (a, b, and
c), and at 1 h (d, e, and f) and 3 h (g, h, and i) after the
temperature downshift to 15°C. Total-cell extracts were analyzed by
2D gel electrophoresis, and autoradiograms were obtained. Only a
portion corresponding to low-molecular-weight proteins is shown. The
cold shock proteins are indicated: 1, CspA; 2, CspB; 3, CspG; 4, CspI.
(B) The amounts of the four proteins were quantitated with a
phosphorimager, with ribosomal protein L11 as a reference. The amounts
of the proteins at each time point upon cold shock are given as the
ratio to the amount of the L11 spot. Since the CspA and CspG spots are
not separated, we counted them together. The ratio of the amount of
protein at 1 h upon cold shock to that at 3 h upon cold shock
is its repression rate. The derepression rate is the ratio of
repression rate in control cells to that in the 5'-UTR overexpression
cells. Lanes: 1, control cells; 2, cells overproducing cspA
5'-UTR; 3, cells overproducing cspI 5'-UTR.
|
|
Concluding remarks.
E. coli has nine csp
genes, cspA to cspI (33). Of these,
cspA (10), cspB (19),
cspG (23), and cspI (this study) are cold shock inducible. All these csp genes share several
important features. (i) They all contain a UP element immediately
upstream of the promoter, which contributes to maintain the high
promoter activity even at low temperatures (9, 22). (ii)
They all contain a long 5'-UTR in their mRNAs (159, 161, 156, and 145 bases for cspA, cspB, cspG, and
cspI, respectively). As found for the cspA mRNA
(22), these 5'-UTRs are believed to exert a negative effect
on their expression at 37°C, while they cause a positive effect on
their cold shock inducibility. (iii) They all have the cold box at the
5'-end region of their 5'-UTRs, which plays a role in autoregulation to
repress their own gene expression at the end of the acclimation phase
(6, 14). (iv) They all contain a downstream box downstream
of the translation initiation codon, which plays an essential
role in the cold shock induction by enhancing translation
(22). Taken together, expression of all four csp genes appears to be regulated essentially in the same manner. It should be mentioned, however, that the optimal temperature ranges
for the induction of these genes are different. CspI is induced
at a relatively lower temperature range than the others.
How are the differences in the temperature range of induction achieved?
It is unlikely that the difference in optimal temperature
is caused at
the level of transcription initiation, because the
putative promoter
region and the UP element are very well conserved
among
cspA,
cspB,
cspG, and
cspI,
as shown in Fig.
1B. It is also
unlikely that it is caused at the
level of translation initiation,
because their Shine-Dalgarno
sequences, downstream boxes, and
coding regions are also quite well
conserved (see Fig.
1B). In
contrast, the nucleotide sequences of their
5'-UTRs are different,
suggesting differences in their mRNA secondary
structures and
therefore in their stabilities. Thus, the 5'-UTRs of the
csp genes
exert a negative effect on their expression at
37°C, affecting
their mRNA stabilities and translation initiation
efficiencies
(
22,
36). These are likely to be differently
modulated upon
temperature downshift, depending upon the secondary
structure
of each mRNA. A larger temperature difference might be
required
for
cspI expression, while a smaller temperature
difference would
be enough for
cspA expression.
It is interesting that although the
cspA gene was
dispensable for cell growth at both high and low temperatures, the
production
of CspB and CspG significantly increased in a
cspA deletion mutant
(
2). This indicates that the
CspA function may be at least
partially complemented by CspB and CspG.
It has been reported
that in
Bacillus subtilis, which
contains three
csp genes, at
least one of the
three
csp genes is required for cell growth (
12).
It seems likely that the functions of CspA, CspB, CspG, and CspI
overlap. To examine this possibility, we are attempting to construct
multiple-deletion strains including a quadruple-deletion mutant.
This approach may reveal a possibility that CspA, CspB, CspG,
and
CspI each play their own specific roles in the
cells.
| |
ACKNOWLEDGMENTS |
We thank R. M. Simons for plasmids. We also thank S. Phadtare for comments.
This work was supported by a grant (to M.I.) from the National
Institutes of Health (GM19043).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Robert Wood Johnson Medical School, University of
Medicine and Dentistry of New Jersey, 675 Hoes Ln., Piscataway, NJ
08854. Phone: (732) 235-4115. Fax: (732) 235-4559. E-mail:
inouye{at}umdnj.edu.
| |
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Journal of Bacteriology, March 1999, p. 1603-1609, Vol. 181, No. 5
0021-9193/99/$04.00+0
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Top
Abstract
Introduction
