UP12 belongs to a family of universal stress proteins UspA family, of which UspA itself, and three additional paralogues, have been characterized previously.. Therefore, we suggest that
Trang 1Identification and characterization of the Escherichia coli stress
Elena S Bochkareva, Alexander S Girshovich and Eitan Bibi
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel
Many groups of proteins play important roles in the cell’s
response to various stresses The molecular chaperone
GroEL of Escherichia coli represents one such highly
con-served family of stress proteins We have obcon-served that
iso-lated GroEL complexes from stationary cultures contain
various polypeptides that can be released from the
chap-eronin by GroES and/or ATP, and identified two such
polypeptides as the proteins GatY and UP12 Whereas
GatY had been isolated previously, as an in vivo substrate of
GroEL, the isolation of UP12 in a complex with GroEL was
intriguing, because based on sequence similarity it was
sug-gested that UP12 might also be a functional stress protein
UP12 belongs to a family of universal stress proteins
(UspA family), of which UspA itself, and three additional
paralogues, have been characterized previously Here we
show that UP12 accumulates under various growth inhibi-tory conditions and induced by heat shock Furthermore, unlike wild-type cells, a UP12 deletion mutant recovers slowly from late stationary growth conditions, and has a marked sensitivity to the toxic agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP) Finally, coimmuno-precipitation experiments confirmed the initial observation that UP12 interacts with GroEL Therefore, we suggest that UP12 may function as a universal stress protein, interaction
of which with GroEL possibly ensures its proper folding state
Keywords: GroEL substrate; UP12; universal stress protein; Stress response; E.coli
Escherichia coli cells undergo a transition from a rapid
growth phase to a stationary phase, which is accompanied
by a variety of physiological changes that affect gene
expression, the structure and composition of the cell wall,
DNA organization, synthesis of storage compounds such as
glycogen and polyphosphate, and other cellular processes
[1,2] As a result of these changes, the cells become resistant
to various deleterious stresses such as heat shock, UV
irradiation, acidic or basic conditions, osmotic shock, and
oxidation [3–5]
Studies carried out in several laboratories have identified
specific cellular networks and individual genes expressed in
the stationary growth phase that improve the survival of
E.coli during prolonged periods of starvation and other
stress conditions [6–11] One of these genes is uspA, which
encodes a small cytoplasmic protein, UspA (universal stress
protein A) that is unique in its universal responsiveness to
diverse stresses [12] The synthesis of UspA is greatly
increased under growth inhibitory conditions, including the
depletion of essential nutrients or exposure to various toxic
agents Moreover, E.coli carrying an inactivated uspA is
more sensitive to prolonged growth inhibition caused by a
variety of starvation and other stress conditions [13,14]
In the course of systematically analyzing the sequenced E.coligenome [15], it has been found that five ORFs share some homologies with UspA Two of them, encoded by ybdQ and ynaF, were previously identified as unknown proteins (UP12 and UP03, respectively) by 2D-PAGE [16] Three E.coli paralogues of UspA have been characterized recently [17], and the results of this study showed that UspA
is a prototype for a family of conserved proteins (universal stress proteins) found not only in bacteria but also in other organisms
Other groups of proteins also play important roles in bacterial stress response One important group includes the heat-shock proteins, whose induction under stress conditions
in E.coli requires the heat-shock transcription factor r32 (rpoH gene product) [18] Many heat-shock proteins, such as members of the Hsp70 and Hsp60 protein families, are molecular chaperones Functionally, they bind to non-native structural forms of various polypeptides and assist them in reaching a native conformation [19] Consequently, as molecular chaperones, they prevent misfolding and aggre-gation of unfolded proteins under heat-shock and other stress conditions [20,21] The E.coli heat-shock protein GroEL belongs to the highly conserved Hsp60 family of oligomeric molecular chaperones named chaperonins [22] GroEL and its small cohort GroES were found to be essential not only under stress, but also for growth under all experimental conditions tested to date [23] GroEL transi-ently interacts (in a GroES- and MgATP-dependent manner) with many unfolded newly synthesized proteins in vitro and
in vivo[24–26] Among the proposed physiological substrates
of GroEL are structurally unstable proteins that require GroEL for permanent conformational maintenance [27]
In the course of GroEL purification from stationary cultures of E.coli, we noticed that a few polypeptides
Correspondence to E Bochkareva, Department of Biological
Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Fax: + 972 89 344118, Tel.: + 972 89 342912,
E-mail: elena.bochkareva@weizmann.ac.il
Abbreviations: CCCP, carbonyl cyanide m-chlorophenyl hydrazone;
DNP, a-dinitrophenol; DM, n-dodecyl-b, D -maltoside; IPTG,
isopro-pyl b- D -thiogalactopyranoside.
(Received 15 February 2002, revised 25 April 2002,
accepted 3 May 2002)
Trang 2consistently co-sedimented with GroEL during sucrose
gradient ultracentrifugation After incubation with GroES
and/or ATP, these polypeptides were released from the
chaperonin One such protein that co-purified with GroEL
was identified as UP12 Based on limited sequence similarity
with UspA, we examined the possibility that UP12 itself
might be a stress protein Here, we show that UP12 interacts
specifically with GroEL, and the results suggest that it plays
a role in the bacterial stress response The possible
physiological relevance of UP12¢s interaction with GroEL
is discussed
E X P E R I M E N T A L P R O C E D U R E S
Bacterial strains and growth conditions
The E.coli strains used in this work are MC4100
[F–araD139D(argF-lac)U169 rpsL150 relA1 flb5301 deoC1
ptsF25 rbsR], TG2 [F¢ traD36 lacIqD(lacZ)M15 supE hsd D5
thiD(lac) proAB) D(srl-recA)306::Tn10 (tetr)], BL21(DE3)
[28] and BW25113 [29] Cultures were grown aerobically in
liquid Luria–Bertani medium with ampicillin, 50 lgÆmL)1
or kanamycin, 30 lgÆmL)1, when necessary For carbon
starvation, cells were grown in M9 minimal medium
with-out amino acids [30], supplemented by a very low glucose
concentration (0.02%) compared with the usual
concentra-tion of 0.4% For phosphate starvaconcentra-tion, cells were grown in
TrisG medium [31] without amino acids supplementation
and with a limited concentration of KH2PO4(0.06 mM) As
a control, cells were grown with the normal concentration of
1.32 mMKH2PO4 Other growth conditions are described
below
Isolation of GroEL and GroEL complexes
from cell extracts
E.coli TG2 bearing plasmid pOA encoding GroEL and
GroES [32] were grown at 37C for 18–20 h in rich 2TY
medium containing ampicillin Typically, 0.5 L of culture
was harvested and the pellet was resuspended in 7 mL of
buffer containing 30 mMTris/HCI (pH 7.5), 60 mMKCI,
10 mM MgCI2, 0.2 mM EDTA, 1 mM dithiothreitol, and
0.5 mM phenylmethanesulfonyl fluoride Cells were then
disrupted by treatment with lysozyme (1 mgÆmL)1) on ice
for 10 min followed by a single rapid cycle of freeze and
thaw at 4C The entire cell lysate was then subjected to a
preparative sucrose gradient centrifugation as follows Each
quarter of the lysate was loaded on top of a 36-mL 10–25%
sucrose gradient prepared in buffer A [40 mM
triethanol-amine-acetate (pH 7.5), 80 mMNH4Cl, 20 mMKCl, 10 mM
MgCl2, 0.1 mMEDTA and 1 mMdithiothreitol] After 21 h
centrifugation at 4C (Beckman L8 ultracentrifuge, SW 27
rotor, 104 000 g) the middle fractions containing GroEL
were pooled and precipitated with ammonium sulfate The
pellet was solubilized in buffer A and subjected to an
additional round of a preparative sucrose gradient
centrif-ugation in buffer B (same as buffer A, but with 450 mM
NH4Cl instead of 80 mM) The GroEL-containing fractions
were pooled, diluted four times in buffer A and
concentra-ted using centriprep 30 (Amicon) The final GroEL
concentration was approximately 10 mgÆmL)1; aliquots
were frozen and stored at)80 C Analytical
ultracentrif-ugations were carried out using 1.4 mL of a linear 5–20%
sucrose gradient in buffer A for 140 min at 4C (Beckman TL100 centrifuge, TLS55 rotor, 250 000 g) GroES was purified as described previously [33]
The dissociation of polypeptides from their complex with GroEL was tested by incubation (30 min at 25C) of the GroEL preparation in buffer A containing 8 mMATP, with
or without GroES (GroES/GroEL¼ 0.2 : 1, w/w) or 0.1% n-dodecyl-b-D-maltoside (DM), followed by centrifugation through a 5–20% sucrose gradient, as described above The top fractions containing free polypeptides were collected, concentrated (using Centricon 30, Amicon) or precipitated
by 10% trichloroacetic acid Samples were subjected to SDS/PAGE and electroblotting on poly(vinylidene difluo-ride) membranes (Bio-Rad) The membranes were then stained for 0.5–1.0 min in 0.04% Coomassie R250 (pre-pared in a solution of 50% methanol and 10% acetic acid) Destaining was carried out in 50% methanol for 5–10 min followed by an extensive wash with water The appropriate bands that corresponded to polypeptides X and Y were excised and subjected to microsequencing analysis (Applied Biosystems Procise Sequencer)
Immunoprecipitation of GroEL complexes from cell extracts was carried out at 4C for 3 h in buffer A, containing 0.01% DM, using protein A–Sepharose pre-loaded with affinity-purified anti-GroEL Ig Blocking of unspecific binding sites was accomplished by incubation with BSA After extensive washing with the same buffer, proteins were eluted from the resin with buffer C [0.1MTris/ HCI (pH 8.0), 1% SDS, 2 mM EDTA and 20 mM
dithiothreitol] and analyzed by SDS/PAGE and Western blotting
Subcloning and deletion of the chromosomal ybdQ gene (encoding UP12)
The chromosomal ybdQ gene encoding UP12 (locus AE000166, accession no U00096 [15]), was amplified by PCR using a 5¢ complementary deoxyoligonucleotide (5¢-CGCGGATCCATGTATAAGACAATCATTATGC-3¢) containing a BamHI site (underlined nucleotides) and a 3¢ deoxyoligonucleotide harboring a HindIII site (5¢-CCCAA GCTTTTAACGCACAACCAGCACC-3¢) as primers with E.coli genomic DNA as a template and Taq polymerase (Roche Molecular Biochemicals) Next, the purified PCR product was ligated with plasmid pGEM-T Easy Vector (Promega) E.coli HB101 cells were transformed with the ligation mixture and a plasmid containing the ybdQ gene insert was isolated The BamHI–HindIII ybdQ fragment was then isolated and subcloned into plasmid pET28a (Novagen), which was digested with the same enzymes The resulting plasmid (pET28yQ) was isolated from E.coli HB101 and the identity of the ybdQ insert was confirmed by DNA sequencing Finally, E.coli BL21(DE3)pLysS was transformed with pET28yQ to overexpress UP12 as a hybrid with an N-terminal extension containing a His6tag separated from UP12 by a thrombin recognition site and two flanking unrelated short sequences The DNA sequen-cing and the deoxyoligonucleotide synthesis were performed
by the Scientific Services Department of the Weizmann Institute of Science
Construction of ybdQ deletion E.coli mutant was carried out as described previously [29] A PCR product was generated by using two 60-mer primers comprised of
Trang 340 nucleotides homologous to regions adjacent to the
beginning and the end of ybdQ and additional 20-nucleotide
complementary to the template plasmid pKD13 carrying
the kanamycin resistance (kan) gene The resulted linear
1.4-kb PCR fragment was gel-purified and introduced by
electroporation into E.coli cells BW25113 containing the
helper plasmid pKD46 Transformants were incubated 1 h
at 37C and overnight at room temperature in SOC
medium and then plated on Luria–Bertani agar plates
containing kanamycin Kanamycin resistant (KmR)
trans-formants were selected and colony-purified on agar plates
incubated overnight at 37C Mutant and wild-type cells
from single colonies were grown overnight in Luria–Bertani
broth at 37C without an antibiotic and tested for loss of
the helper plasmid pKD46 The correct chromosomal
structure of DybdQ::kan mutant was verified by three
independent PCR experiments which were carried out with
two 20-nucleotide primers complementary to regions
flank-ing the ybdQ gene as a pair and in combination with two
kan– specific primers (k2 and kt; [29]) An additional PCR
experiment with the two primers used for subcloning of
ybdQ(see above), did not yield any product, as expected
Finally, UP12 expression in the mutant and wild-type cells
was examined by Western blotting, as described below
Purification of UP12 and preparation of anti-UP12 Ig
The UP12 hybrid containing a His6 tag (see above) was
purified from E.coli BL21(DE3)pLysS(pET28yQ) Briefly,
cultures were grown in 2TY medium at 37C and induced
with isopropyl thio-b-D-galactoside (IPTG; 1 mM) at the
exponential growth phase (D600¼ 0.4) for 2.5 h After
centrifugation, the cells were resuspended in 7.5 mL of
buffer K [20 mMTris/HCI (pH 8.0) and 0.5MNaCl] and
disrupted by sonication using Microson (Heat Systems,
Inc.) The tagged UP12 was purified from the cell extract by
affinity chromatography using His-bind resin (Novagen),
according to the manufacturer’s instructions The protein
was eluted with buffer K containing 1M imidazole and
cleaved by thrombin (Novagen) at room temperature for
17–20 h, using a protease/protein ratio of 1 : 800 The
protein concentration was measured using a Bradford
solution (Bio-Rad) and BSA as a standard Polyclonal
anti-UP12 Ig were produced in rabbits by the Scientific Services
Department of the Weizmann Institute by a single injection
of 150 lg of the purified protein, followed by two booster
shots of the same amount of protein at 2-week intervals
Serum was collected and used for immunoblotting at a
1 : 5000 dilution
SDS/PAGE and Western blotting
In order to estimate the intracellular concentration of UP12
under various conditions, we centrifuged culture samples
containing equal amounts of cells (corresponding to
D600¼ 0.8) The pellets were washed with 0.2 mL of 10%
sucrose prepared in 10 mM Tris/HCI (pH 8.0) and then
lysed by adding 70 lL of buffer C (see above) containing
0.1 mMdithiothreitol instead of 20 mM After incubation at
room temperature for 10 min, the lysates were centrifuged
to remove the pelleted DNA, and the protein concentrations
in the supernatants were measured using a Bio-Rad DC
protein assay The supernatants were diluted 1 : 2 by
solution D (solution C with 20% glycerol, 0.002% bromo-phenol blue and 30 mMdithiothreitol) and then incubated
at 86C for 8 min Typically, cell extracts (5–10 lg of protein) were separated by SDS/PAGE using the standard Laemmli system [30] with 13% and 4% (w/w) acrylamide in the separating and stacking gels, respectively In order to estimate the ratio between the amounts of UP12 and the total amount of protein in the extracts, a series of samples containing determined amounts of the purified UP12 were separated by SDS/PAGE along with the cell extract samples Immunoblots were performed according to the ECL Western blotting protocol (Amersham), and the chemiluminescence was detected by exposure of the mem-branes to films Protein quantities were estimated by scan-ning densitometry using a Bio-Rad Imaging Densitometer (Model GS-690)
R E S U L T S
Isolation of GroEL-polypeptide complexes from a stationary-phase culture
The chaperonin GroEL comprises 14 identical subunits of 57.3 kDa and has a unique molecular mass of 800 kDa that can be separated from almost all other E.coli proteins
by sucrose gradient centrifugation [19,24] In order to isolate GroEL accompanied by cytoplasmic proteins from station-ary cultures, we prepared cell extracts from 20-h cultures of E.coli TG2(pOA) The extracts were subjected to three successive steps of sucrose gradient centrifugation We noticed that a few polypeptides consistently cosedimented with GroEL and were found exclusively in the GroEL-containing fractions after a third round of sucrose gradient centrifugation (Fig 1A), suggesting that these polypeptides might be in vivo substrates of GroEL Therefore, we analyzed the effect of ATP and GroES on their dissociation
Fig 1 ATP-dependent cosedimentation of proteins with chaperonin GroEL (A) Detection of polypeptides in the GroEL-containing frac-tions of sucrose gradient The crude preparation of GroEL (100 lg) was subjected to analytical sucrose gradient centrifugation, as des-cribed in Experimental procedures Proteins in 14 fractions (100 lL each) collected from the top of gradient were precipitated by TCA, separated by SDS/PAGE and visualized by Coomassie staining Lane
15 contains protein molecular mass markers (B) The effect of ATP preincubation on releasing of polypeptides from GroEL Before sucrose gradient centrifugation the GroEL preparation (40 lg) was treated with 8 m M ATP (lane 1) or 8 m M ATP in the presence of 0.1%
DM (lanes 2–4) for 30 min at 25 C Fractions (300 lL) was collected, proceeded as in (A) and only some of them are presented Lanes 1 and
2, as fractions 1–3 in (A), contain proteins recovered from the top of sucrose gradient; lane 3 is intermediate fraction; and lane 4 corres-ponding to fractions 7–10 in (A) contains oligomeric GroEL.
Trang 4from GroEL As shown in Fig 1B, a 16-kDa polypeptide
(Y) dissociates from GroEL by treatment with ATP,
whereas other polypeptides, including a 30-kDa polypeptide
(X) require both ATP and the co-chaperonin GroES for
dissociation (data not shown) Interestingly, the nonionic
detergent DM mimicked the effect of GroES by releasing
almost all of the polypeptides from GroEL After treatment
with ATP and 0.1% DM, these polypeptides were recovered
in the top three fractions of the sucrose gradient (Fig 1B,
lane 2) The ATP/GroES-promoted dissociation of these
polypeptides from GroEL, as well as the mild conditions
under which the cell extracts were prepared (see
Experi-mental procedures) suggest that the isolated GroEL
com-plexes may represent true physiologically relevant
interactions in stationarily grown cells
Identification of proteins X (GatY) and Y (UP12)
In order to identify polypeptides X and Y, we repeated the
experiment (Fig 1B) on a preparative scale Briefly, after
their dissociation from the GroEL complex, proteins were
separated by SDS/PAGE, electroblotted onto
poly(vinylid-ene difluoride) membranes, and subjected to N-terminal
sequencing The amino-acid sequences of proteins X and Y
were identified in the Swiss-Prot databank as GatY and
UP12, respectively GatY (D-tagatose-1,6-bis-phosphate
aldolase of class II), is a homotetrameric protein consisting
of 31-kDa subunits; it belongs to a family of lyases involved
in carbohydrate metabolism GatY is highly thermolabile
and is degraded in vivo at temperatures above 30C [34]
Our results are in agreement with those of a recent work in
which GatY has been identified by other means as an in vivo
substrate of GroEL [27] Interestingly, GatY is also included
in a list of proteins that aggregate at 42C in E.coli
containing a dnaK deletion mutation (DnaK is a Hsp70
chaperone) [35] Collectively, our observations and those of
others suggest that folding of GatY might require the
assistance of molecular chaperones, which bind to its
temperature-induced flexible conformation
The second protein that was isolated in a complex with
GroEL was the 16-kDa protein UP12 (also termed UspG
[17]) This protein is encoded by the ybdQ gene and belongs
to the UPF0022 (UspA) protein family [15] E.coli contains
five small members of this family including UspA itself
(Fig 2), and one larger protein consisting of two UspA
domains in tandem [17] The small members of this family
are proteins of 142–144 amino-acids long; they are acidic
and presumably located in the cytoplasm like UspA
Members of this family share a strikingly similar hydro-pathy profile (data not shown), and UP12 shares 27% identical and similar residues with UspA Taken together, although the sequence similarity between UP12 and UspA
is not very pronounced, the following observations support the classification of UP12 as a member of the UspA family
Sub-cloning, purification, and characterization of UP12 and its interaction with GroEL
In order to investigate the suggestion that UP12 is a functional member of the universal stress protein family and further characterize its interaction with GroEL, we cloned the UP12 encoding gene (ybdQ) by PCR YbdQ was inserted into the expression vector pET-28a, under the control of the T7 promoter A UP12 hybrid protein of 21 kDa containing
an N-terminal His6 tag was overexpressed, purified by nickel-affinity chromatography, and treated with thrombin (Fig 3A) The resulting cleaved UP12 hybrid, which contains a 15-residue N-terminal extension (Fig 3A, lane 6), was used to raise antibodies in rabbits Western blotting revealed that the anti-UP12 Ig recognizes the two hybrid forms of the isolated protein (before and after cleavage with thrombin; Fig 3B, lane 1) In addition, Western blotting of the total E.coli extracts demonstrated that the antibodies selectively recognize a 16-kDa protein that corresponds to UP12 (Fig 3B, lane 2)
In order to investigate whether UP12 interacts with GroEL in vivo, we analyzed GroEL complexes by co-immunoprecipitation GroEL complexes were isolated from late stationary cultures of E.coli TG2(pOA) overexpressing GroEL by immunoprecipitation with anti-GroEL Ig As shown in Fig 3C, 35% of the UP12 were co-immuno-precipitated with the GroEL Remarkably, upon treatment
of the extracts with ATP, UP12 is completely released from the GroEL complex (Fig 3C, lanes 2 and 4) When extracts prepared from late stationary cultures of E.coli MC4100 or TG2 that do not overexpress GroEL were subjected to a similar analysis, 4–5% of UP12 was found in a complex with GroEL (data not shown) Similar yields were obtained previously for some of in vivo GroEL substrates isolated from the exponentially grown cells by immunoprecipitation with anti-GroEL Ig [27] The high yield of the UP12-GroEL complex isolation and the ATP-mediated dissociation of the complex strongly support the suggestion that the two stress proteins GroEL and UP12 interact with each other
in vivo, and that this interaction might be physiologically relevant
Fig 2 Sequence alignment of members of the E coli UspA family Optimal alignment of the best regions of similarity among the sequences was performed using the program PRETTYBOX (Wisconsin GCG package, Version 10) A black or a gray background indicates identical and similar residues, respectively Swiss-Prot accession numbers are as follows: UspA, P28242 [12], YiiT, P32163; UP03, P37903; UP12, P39177; YecG, P46888 [15].
Trang 5UP12 is highly expressed at the stationary phase and
under conditions of phosphate or carbon starvation
To explore the properties of UP12 as a possible stress
protein, we analyzed the level of UP12 expression in E.coli
cells cultured in various media and under different
experi-mental conditions Cultures of E.coli MC4100 were grown
at 37C in Luria–Bertani, and samples were withdrawn at
the indicated times (Fig 4A) Cell extracts were then
subjected to SDS/PAGE and electroblotting, and the
relative amount of UP12 in each sample was estimated by
semiquantitative Western blotting (Fig 4B) As shown,
UP12 is hardly expressed during exponential growth; it
starts to accumulate at the early stationary phase, and its
steady-state level increases further during the late stationary phase As a result of this accumulation, the relative amount
of UP12 in stationary cells is about 10 times higher than that observed at the beginning of growth (Fig 4A) For comparison, the amount of GroEL was determined in the same extracts using anti-GroEL Ig It has been previously shown that at the beginning of the stationary phase the rate
of synthesis of heat-shock proteins increases considerably, but only transiently [2,18] Similarly, we observed that the steady-state amount of GroEL remains constant during growth, with only a slight increase ( twofold) at the stationary phase (Fig 4B)
As shown, UP12 accumulates in cells grown in Luria– Bertani during the stationary growth phase, possibly due to nutrient exhaustion In order to examine whether UP12 is induced by starvation, we tested the expression of UP12 in cells grown in minimal media containing limited concen-trations of phosphate or a carbon source The amount of UP12 increased dramatically under both starvation condi-tions (Fig 4C) With phosphate starvation, UP12 accumu-lation follows the arrest of growth, whereas under carbon starvation conditions an 1 h delay is observed (Fig 4C) Interestingly, in supplemented minimal media, unlike in Luria–Bertani, UP12 accumulation occurs only after pro-longed (10–15 h) incubation of growth ceasing cells (Fig 4C and data not shown) Taken together, our results indicate that the accumulation of UP12 is not due to a certain stress caused by exhausting a specific nutrient in the medium, but rather as a result of general growth inhibitory conditions
UP12 expression is induced in response to toxic agents and increased temperature
Next, we examined the effect of toxic agents on UP12 expression As shown in Fig 5, the addition of DNP or CCCP to exponential cultures led to an immediate arrest of growth followed by an increased expression of UP12 In the presence of DNP, the induction of UP12 was somewhat slower compared with the rapid response to CCCP In both cases, however, after prolonged incubation with the toxic compounds (4–5 h), the steady-state amount of UP12 increased up to fivefold its amount in untreated cells (Fig 5A,B) In order to test the expression of UP12 under cold or heat shock conditions, cultures were grown at 37C
in Luria–Bertani, and then transferred at the mid-log phase
to either 30C or 44 C As shown in Fig 5C, the rate by which UP12 expression was increased at 30C is similar to that at 37C In contrast, heat shock at 44 C induced a remarkably rapid accumulation of UP12 Therefore, increased synthesis of UP12 occurs not only in growth-arrested cells but also under heat shock conditions
TheE coli DybdQ mutant shows a reduced growth rate during stationary-phase-exit and an increased sensitivity
to CCCP
In order to study the possible biological function of UP12, an E.colimutant deleted of the UP12 encoding gene (ybdQ) was explored The mutated DybdQ::kan strain was constructed as described previously [29] with the E.coli strain BW25113 This strain behaves as E.coli MC4100 or TG2, with regard
to its UP12 expression pattern (data not shown), and as
Fig 3 Purification of His 6 –UP12, characterization of the anti-UP12 Ig
and coimmunoprecipitation of UP12 with GroEL from cell extracts (A)
Purification of UP12 His 6 –UP12 as a hybrid with an N-terminal
extension containing a His 6 tag separated from UP12 by a thrombin
recognition site and two unrelated short sequences was purified from
E.coli BL21(DE3)pLysS cells harboring pET28yQ by affinity
chro-matography on His-bind resin, as described in Experimental
proce-dures Fractions of 15 mL of washing solution and 2.5 mL of eluates
were collected and 0.15% of each fraction was subjected to SDS/
PAGE After electrophoresis the gel was stained with Coomassie Blue.
Lane 1, total cell extract; lanes 2, 3 and 4, column wash fractions (with
5 m M , 60 m M and 90 m M imidazole, respectively); lane 5, elution
fraction (with 1 M imidazole); lane 6, purified His 6 -UP12 after cleavage
by thrombin Lane 7 contains protein markers Arrow indicates the
position of a hybrid protein, His 6 –UP12 (B) Western blotting with
anti-UP12 Ig Lane 1, purified His 6 –UP12 (10 ng) after incomplete
cleavage with thrombin; lane 2, total cell extract (5 lg protein)
pre-pared from E.coli MC4100 grown in Luria–Bertani medium for 24 h
at 37 C Arrow indicates the position of UP12 (C)
Co-immunopre-cipitation of UP12 with GroEL from cell extracts Isolation of GroEL
complexes from the E.coli TG2(pOA) cell extracts using protein
A–Sepharose preloaded with affinity-purified anti-GroEL Ig were
performed as described in Experimental procedures UP12
coimmu-noprecipitated with GroEL from 15 and 30 lg of the cell lysate without
(lanes 1 and 3) or in the presence of ATP (lanes 2 and 4) was detected
with anti-UP12 Ig Samples (3 and 6 lg of total proteins) of the cell
extract that was not immunoprecipitated are shown in lanes 5 and 6.
Trang 6expected, the deletion mutant does not express UP12
(Fig 6A,B, insets) The ability of the mutant strain to
resume growth after the stationary phase was then compared
with that of the parental strain As shown in Fig 6A, after transfer from stationary cultures into fresh Luria–Bertani broth, both strains reached almost the same D600 value
Fig 4 UP12 expression is induced under growth inhibitory conditions at the stationary phase and as a result of starvation E.coli MC4100 was grown
at 37 C in Luria–Bertani or minimal media and the samples were withdrawn at 30-min intervals The cell density in each sample was measured
by absorption at 550 or 420 nm Equal amounts of cells were collected from each sample, lysed by SDS-buffer, and UP12 amount in samples was estimated by Western blotting (A) Accumulation of UP12 at the stationary phase during growth of cells in Luria–Bertani Absorption (s) and relative amount of UP12 (d) were measured for 12 samples of cells taken at the indicated time (B) Western blot analysis of proteins in samples collected in (A) 5 lg and 1 lg of total protein in samples were separated by SDS/PAGE and immunoblotted with anti-UP12 Ig and anti-GroEL Ig, respectively Quantification of protein bands was performed by scanning densitometry, as described in Experimental procedures (C) Effect of phosphate or carbon starvation on cell growth and expression of UP12 Absorption (upper panel) and relative amount of UP12 (lower panel) in samples of cells grown in minimal M9 medium containing limited concentration of glucose (m) or in TrisG medium with limited (d) and normal (s) phosphate concentration The growth arrest start point is indicated by a perpendicular dashed line.
Fig 5 Effect of toxic agents and
tempera-ture shift on UP12 expression Exponential
cultures of E.coli MC4100 grown at 37 C
in complete TrisG medium were exposed to
toxic agents at the indicated time (designated
zero) Alternatively, three cultures of cells
were grown at 37 C in Luria–Bertani At
D 550 ¼ 0.6, two of the cultures were
trans-ferred from 37 C to 30 C or to 44 C The
third culture was left at 37 C Samples of
cells were withdrawn at 10–30 min intervals
and after measuring the D 550 value, cells
were lysed by SDS-containing solution the
relative amount of UP12 in the samples and
was estimated, as described in the legend to
Fig 4 (A) Effect of addition of 4 m M DNP
on cell growth (h) and expression of UP12
(j) (B) Monitoring of cell growth (s) and
amount of UP12 (d) before and after
addition of 0.1 m M CCCP (C) Effect of
temperature on cell growth (left panel) and
UP12 accumulation (right panel).
Trang 7However, the mutated strain exhibits a reduced growth rate
compared with the isogenic wild-type strain The generation
time of the mutant soon after the transfer to fresh medium is
63 min, which is 1.5-fold higher than that of the wild-type
(42 min) Similarly, after prolonged growth (20–24 h) in
phosphate-supplemented minimal medium, the mutant
reproducibly demonstrated a marked recovery lag when
transferred to a phosphate-limited medium, whereas the
wild-type cells recovered rapidly (Fig 6B) Therefore, we
propose that UP12 plays a role during the recovery of E.coli
from the stationary phase As UP12 expression is induced by
treatment with toxic compounds such as DNP and CCCP
(Fig 5), we investigated the sensitivity of E.coli DybdQ::km
to the toxic agents As shown in Fig 6C, the mutant exhibits
an increased sensitivity to CCCP compared with the parental
strain In conclusion, the phenotype of the UP12-deletion
strain provides additional support to the suggestion that
UP12 is a stress protein
D I S C U S S I O N
In this work, we have identified two proteins GatY and
UP12 as putative in vivo substrates of the chaperonin
GroEL In addition to its in vivo interaction with GroEL
[27], it was shown that GatY aggregates at 42C in mutant
cells containing a deletion for DnaK [35] Taken together, these observations indicate that maintenance of the correct folding state of GatY in E.coli probably requires the assistance of two chaperone systems The identification of UP12 as a putative in vivo substrate of GroEL was interesting, because this protein belongs to a family of universal stress proteins (UspA family) As shown previ-ously [12,14,17], the synthesis of UspA and three of its paralogues is greatly increased under various stress condi-tions that cause the arrest of growth In addition, a temperature shift from 28 to 42C resulted in a several-fold induction of UspA expression [13] A mutant strain lacking UspA exhibits an enhanced sensitivity to several toxic agents and a reduced ability to survive prolonged carbon starvation Based on these and other results, it has been suggested that UspA has a general protection function
in growth-arrested E.coli cells [13]
In this work we characterized UP12, a member of the UspA family, and showed that the expression pattern of UP12 under starvation, heat shock, and other stress condi-tions is not identical but is similar to that of UspA In addition, we found that some properties of a mutant deleted
of the UP12 encoding gene resemble those of the uspA-deleted mutant Therefore, we suggest that UP12 is also a stress protein Unlike UspA, however, the properties of
Fig 6 Effect of ybdQ deletion on cell growth at 37 °C and on sensitivity towards CCCP exposure (A) Growth curves of wild-type and D ybdQ cells in Luria–Bertani broth Single colonies of wild-type and D ybdQ strains were plated on Luria–Bertani agar plates After overnight incubation, several colonies of each strain were suspended in Luria–Bertani broth, diluted to the same density (D 600 0.030) in flasks with Luria–Bertani, and growth was followed by D 600 measurements Inset: detection of UP12 in cell extracts prepared from wild-type (WT) and D ybdQ (D) cells grown overnight in Luria–Bertani broth by Western blotting (B) Effect of ybdQ deletion on cell growth in minimal TrisG medium with limited phosphate concen-tration Overnight (20 h) cultures of wild-type and DybdQ strains in TrisG medium supplemented with normal phosphate concentration (1.32 m M
KH 2 PO 4 ) were diluted into TrisG with limited phosphate (0.06 m M KH 2 PO 4 ) and the D 420 was followed during growth Inset, Detection of UP12 in the overnight TrisG cultures by Western blotting (C) Sensitivity of wild-type and UP12-depleted E.coli towards CCCP exposure Duplicated cultures of wild-type and D ybdQ strains were grown at 37 C in TrisG medium supplemented with 1.32 m M KH 2 PO 4 until D 420 ¼ 0.6 Serial dilutions of cells were spotted on Luria–Bertani plates supplemented with the indicated concentrations of CCCP and incubated overnight
at 37 C.
Trang 8UP12 do not exactly match the definition of a universal stress
protein For example, in our studies of UP12 expression in
cells treated with various toxic agents, we observed that some
of the compounds that affected UspA expression also
induced the synthesis of UP12 (CCCP and DNP) In
contrast, other toxic compounds, such as H2O2or CdCI2,
at concentrations that affected synthesis of UspA [13] had no
effect on UP12 expression (data not shown) In addition,
although the deletion of the UP12 encoding gene increased
the sensitivity of the mutant to CCCP (Fig 6), it did not have
any detectable effects on the sensitivity to mitomycin C,
unlike in the case of uspA deletion ([14] and our data, not
shown) Consequently, it is possible that under various stress
conditions, UP12 and UspA have distinct, but overlapping
functions In addition, it is also possible that under certain
conditions, the loss of UP12 expression is compensated by
backup systems, such as other members of the UspA family
However, this assumption needs to be examined further In
this regard, the exact mode of action of UspA, as well as that
of other members of the UspA family remains to be explored
According to our findings, it is clear that UP12 expression
is induced under various stress conditions However, it is not
yet known how UP12 expression is regulated at the
molecular level All the E.coli members of the UspA family
are encoded by monocystronic genes dispersed throughout
the chromosome, but unlike many other stress-related
E.coligenes, their promoters are probably recognized by
the housekeeping r70factor [13,15] The control of uspA
expression has been studied extensively, and it has been
shown that it is regulated positively by ppGpp of the
stringent response, RecA of the SOS modulon, and two
members of the CspA family, CspC and CspE [9,14,36,37]
In this regard, it is interesting that three UspA paralogues
are regulated in a similar manner [17]
In the present study, we revealed that UP12 interacts
efficiently with GroEL, as 35% of the steady-state
amount of UP12 was found in a complex with the
chaperonin under certain conditions (Fig 3C) This
inter-action is specific, because UP12 is removed from GroEL
by ATP [38] Furthermore, GroEL seems to exhibit a high
selectivity towards UP12 compared to other proteins of
the UspA family and also in comparison with all other
small cytoplasmic proteins (Mrless than 20 kDa; Fig 1)
The efficient and selective interaction with GroEL might
reflect the UP12 flexible tertiary structure Our preliminary
results indicate that isolated UP12 is very sensitive to
proteolysis suggesting that this protein easily acquires
unstable conformation(s) As a result, UP12 might be
continuously recognized by GroEL Therefore, we suggest
that UP12 is a persistent in vivo GroEL substrate,
although it cannot be excluded that UP12 may co-operate
functionally with GroEL under some stress conditions
Such co-operation might also be important for UP12 to
perform its role during the recovery of E.coli from the
stationary phase
A C K N O W L E D G E M E N T S
This paper is dedicated to the memory of the late Professor Alexander
Girshovich who initiated and was actively involved in the beginning of
this work We wish to thank I Gokhman for her help during the
subcloning of YbdQ This research was supported by the MINERVA
Foundation, Munich/Germany.
R E F E R E N C E S
1 Kolter, R., Siegele, D.A & Tormo, A (1993) The stationary phase
of the bacterial life cycle Annu.Rev.Microbiol.47, 855–874.
2 Huisman, G.W., Siegele, D.A., Zambrano, M.M & Kolter, R (1996) Morphological and physiological changes during stationary phase, In Escherichia Coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F.C., Curtis III, R., Ingraham, J.L., Lin, E.C.C., Low, K.B., Magasanik, B., Reznokoff, W.S., Riley, M., Schaechter, M & Umbarger, H.E., eds) 2nd edn pp 1672–1682 ASM Press, Washington, DC.
3 Jenkins, D.E., Scshultz, J.E & Matin, A (1988) Starvation-induced cross-protection against heat or H 2 O 2 challenge in Escherichia coli J.Bacteriol.170, 3910–3914.
4 Lange, R & Hengge-Aronis, R (1991) Identification of a central regulator of stationary phase gene expression in Escherichia coli Mol.Microbiol.5, 49–59.
5 Small, P., Blankenhorn, D., Welty, D., Zinser, E & Slonczewski, J.L (1994) Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH J.Bacteriol.176, 1729–1737.
6 Jenkins, D.E., Auger, E.A & Matin, A (1991) Role of RpoH, a heat shock regulator protein in Escherichia Coli carbon starvation protein synthesis and survival J.Bacteriol.173, 1992–1996.
7 Li, C., Ichikawa, J.K., Ravetto, J.J., Kuo, H.C., Fu, J.C & Clarke, S (1994) A new gene involved in stationary-phase survival located at 59 minutes on the Escherichia coli chromosome J.Bacteriol.176, 6015–6022.
8 Williams, M.D., Ouyang, T.X & Flickinger, M.C (1994) Star-vation-induced expression of SspA and SspB: the effects of a null mutation in sspA on Escherichia coli protein synthesis and survival during growth and prolonged starvation Mol.Microbiol.11, 1029–1043.
9 Farewell, A., Diez, A.A., DiRusso, C.C & Nystro¨m, T (1996) Role of the Escherichia coli FadR regulator in stasis survival and growth phase-dependent expression of the uspA, fad, and fab genes J.Bacteriol.178, 6443–6450.
10 Siegele, D.A & Guynn, L.J (1996) Escherichia coli proteins syn-thesized during recovery from starvation J.Bacteriol.178, 6352– 6356.
11 Hengge-Aronis, R (1999) Interplay of global regulators and cell physiology in the general stress response of Escherichia coli Curr Opin.Microbiol.2, 148–152.
12 Nystro¨m, T & Neidhardt, F.C (1992) Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein
in Escherichia coli Mol.Microbiol.6, 3187–3198.
13 Nystro¨m, T & Neidhardt, F.C (1994) Expression and role of the universal stress protein, UspA, of Escherichia coli during growth arrest Mol.Microbiol.11, 537–544.
14 Diez, A., Gustavsson, N & Nystro¨m, T (2000) The universal stress protein A of Escherichia coli is required for resistance to DNA damaging agents and is regulated by a RecA/FtsK-dependent regulatory pathway Mol.Microbiol.36, 1494–1503.
15 Blattner, F.R., Plunkett III, G., Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., Rode, C.K., Mayhew, G.F et al (1997) The complete genome sequence
of Escherichia coli K-12 Science 277, 1453–1474.
16 Pasquali, C., Frutiger, S., Wilkins, M.R., Hughes, G.J., Appel, R.D., Bairoch, D., Schaller, A., Sanchez, J.C & Hochstrasser, D.F (1996) Two-dimensional gel electrophoresis of Escherichia coli homogenates: the Escherichia coli SWISS-2D PAGE data-base Electrophoresis 17, 547–555.
17 Gustavsson, N., Diez, A.A & Nystro¨m, T (2002) The universal stress protein paralogues of Escherichia coli are co-ordinately regulated and co-operate in the defence against DNA damage Mol.Microbiol.43, 107–117.
Trang 918 Gross, C.A (1996) Function and regulation of the heat shock
proteins, In Escherichia Coli and Salmonella: Cellular and
Molecular Biology (Neidhardt, F.C., Curtis III, R., Ingraham,
J.L., Lin, E.C.C., Low, K.B., Magasanik, B., Reznokoff, W.S.,
Riley, M., Schaechter, M & Umbarger, H.E., eds) 2nd edn.
pp 1382–1399 ASM Press, Washington, DC.
19 Bukau, B & Horwich, A.L (1998) The Hsp70 and Hsp60
cha-perone machines Cell 92, 351–366.
20 Gething, M.J & Sambrook, J (1992) Protein folding in the cell.
Nature 355, 33–45.
21 Gragerov, A., Nudler, E., Komissarova, N., Gaitanaris, G.A.,
Gottesman, M.E & Nikiforov, V (1992) Cooperation of GroEL/
GroES and DnaK/DnaJ heat shock proteins in preventing protein
misfolding in Escherichia coli Proc.Natl Acad.Sci.USA 89,
10341–10344.
22 Ellis, R.J (1996) Discovery of molecular chaperones Cell Stress
Chaperones 1, 155–160.
23 Fayet, O., Ziegelhoffer, T & Georgopoulos, C (1989) The groES
and groEL heat shock gene products of Escherichia coli are
essential for bacterial growth at all temperatures J.Bacteriol.171,
1379–1385.
24 Bochkareva, E.S., Lissin, N.M & Girshovich, A.S (1988)
Tran-sient association of newly synthesized unfolded proteins with the
heat-shock protein GroEL Nature 336, 254–257.
25 Lorimer, G.H (1996) A quantitative assessment of the role of
the chaperonin proteins in protein folding in vivo FASEB J 10,
5–9.
26 Ewalt, K.L., Hendrick, J.P., Houry, W.A & Hartl, F.U (1997)
In vivo observation of polypeptide flux through the bacterial
chaperonin system Cell 90, 491–500.
27 Houry, W.A., Frishman, D., Eckerskorn, C., Lottspeich, F &
Hartl, F.U (1999) Identification of in vivo substrates of the
cha-peronin GroEL Nature 402, 147–154.
28 Studier, F.W., Rosenberg, A.H., Dunn, J.J & Dudendorff, J.W.
(1990) Use of T7 RNA polymerase to direct expression of cloned
genes Methods Enzymol 185, 60–89.
29 Datsenko, K.A & Wanner, B.L (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products Proc.Natl Acad.Sci.USA 97, 6640–6645.
30 Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
31 Echols, H., Garen, A., Garen, S & Torriani, A (1961) Genetic control of repression of alkaline phosphotase in E.coli J.Mol Biol 3, 425–438.
32 Horovitz, A., Bochkareva, E.S., Kovalenko, O & Girshovich, A.S (1993) Mutation Ala2 fi Ser destabilizes intersubunit inter-actions in the molecular chaperone GroEL J.Mol.Biol.231, 58–64.
33 Kamireddi, M., Eisenstein, E & Reddy, P (1997) Stable expres-sion and rapid purification of Escherichia coli GroEL and GroES chaperonins Prot.Exp.Purif.11, 47–52.
34 Nobelmann, B & Lengeler, J.W (1996) Molecular analysis of the gat genes from Escherichia coli and of their roles in galactitol transport and metabolism J.Bacteriol.178, 6790–6795.
35 Mogk, A., Tomoyasu, T., Goloubinoff, P., Rudiger, S., Roder, D., Langen, H & Bukau, B (1999) Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation
by DnaK and ClpB EMBO J 18, 6934–6949.
36 Kvint, K., Hosbond, C., Farewell, A., Nybroe, O & Nystro¨m, T (2000) Emergency derepression: stringency allows RNA poly-merase to override negative control by an active repressor Mol Microbiol 35, 435–443.
37 Phadtare, S & Inouye, M (2001) Role of CspC and CspE in regulation of expression of RpoS and UspA, the stress response proteins in Escherichia coli J.Bacteriol.183, 1205–1214.
38 Sigler, P.B., Xu, Z., Rye, H.S., Burston, S.G., Fenton, W.A & Horwich, A.L (1998) Structure and function in GroEL-mediated protein folding Annu.Rev.Biochem.67, 581–608.