Tài liệu Báo cáo Y học: Identification and characterization of the Escherichia coli stress protein UP12, a putative in vivo substrate of GroEL pptx

Identification and characterization of the Escherichia coli stress protein UP12, a putative in vivo substrate of GroEL 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 observed 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. coli genome [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 r 32 (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 foundto 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) Eur. J. Biochem. 269, 3032–3040 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02978.x consistently 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. EXPERIMENTAL PROCEDURES 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 lacI q D(lacZ)M15 supE hsd D5 thi D(lac ) proAB) D(srl-recA)306::Tn10 (tet r) ], 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 starvation, cells were grown in TrisG medium [31] without amino acids supplementation and with a limited concentration of KH 2 PO 4 (0.06 m M ). As a control, cells were grown with the normal concentration of 1.32 m M KH 2 PO 4 . 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 37 °C 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 m M Tris/HCI (pH 7.5), 60 m M KCI, 10 m M MgCI 2 ,0.2m M EDTA, 1 m M dithiothreitol, and 0.5 m M phenylmethanesulfonyl fluoride. Cells were then disrupted by treatment with lysozyme (1 mgÆmL )1 )onice for 10 min followed by a single rapid cycle of freeze and thaw at 4 °C. 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 m M triethanol- amine-acetate (pH 7.5), 80 m M NH 4 Cl, 20 m M KCl, 10 m M MgCl 2 ,0.1m M EDTA and 1 m M dithiothreitol]. After 21 h centrifugation at 4 °C (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 m M NH 4 Cl instead of 80 m M ). 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 4 °C (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 25 °C) of the GroEL preparation in buffer A containing 8 m M ATP, with or without GroES (GroES/GroEL ¼ 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 4 °C 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 [0.1 M Tris/ HCI (pH 8.0), 1% SDS, 2 m M EDTA and 20 m M 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 PCRusinga5¢ complementary deoxyoligonucleotide (5¢-CGC GGATCCATGTATAAGACAATCATTATGC-3¢) containing a BamHI site (underlined nucleotides) and a 3¢ deoxyoligonucleotide harboring a HindIII site (5¢-CCC AA 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 His 6 tag 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 Ó FEBS 2002 UP12 is an E. coli universal stress protein (Eur. J. Biochem. 269) 3033 40 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 37 °C and overnight at room temperature in SOC medium and then plated on Luria–Bertani agar plates containing kanamycin. Kanamycin resistant (Km R )trans- formants were selected and colony-purified on agar plates incubated overnight at 37 °C. Mutant and wild-type cells from single colonies were grown overnight in Luria–Bertani broth at 37 °C 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 His 6 tag (see above) was purified from E. coli BL21(DE3)pLysS(pET28yQ). Briefly, cultures were grown in 2TY medium at 37 °C and induced with isopropyl thio-b- D -galactoside (IPTG; 1 m M )atthe exponential growth phase (D 600 ¼ 0.4) for 2.5 h. After centrifugation, the cells were resuspended in 7.5 mL of buffer K [20 m M Tris/HCI (pH 8.0) and 0.5 M NaCl] 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 1 M 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 D 600 ¼ 0.8). The pellets were washed with 0.2 mL of 10% sucrose prepared in 10 m M Tris/HCI (pH 8.0) and then lysed by adding 70 lL of buffer C (see above) containing 0.1 m M dithiothreitol instead of 20 m M . 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 m M dithiothreitol) and then incubated at 86 °C for 8 min. Typically, cell extracts (5–10 lgof 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). RESULTS 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. 3034 E. S. Bochkareva et al. (Eur. J. Biochem. 269) Ó FEBS 2002 from 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 30 °C [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 42 °CinE. 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)byPCR.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 His 6 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 showninFig.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]. Ó FEBS 2002 UP12 is an E. coli universal stress protein (Eur. J. Biochem. 269) 3035 UP12 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 37 °C 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 37 °C in Luria–Bertani, and then transferred at the mid-log phase to either 30 °Cor44°C.AsshowninFig.5C,therateby which UP12 expression was increased at 30 °C is similar to that at 37 °C. 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. The E. 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. coli mutant 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 5m M ,60m 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- noprecipitatedwithGroELfrom15and30 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. 3036 E. S. Bochkareva et al. (Eur. J. Biochem. 269) Ó FEBS 2002 expected, 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 D 600 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 lgand1 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)andnormal (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 °Cto30°Corto44°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). Ó FEBS 2002 UP12 is an E. coli universal stress protein (Eur. J. Biochem. 269) 3037 However, the mutated strain exhibits a reduced growth rate comparedwiththeisogenicwild-typestrain.Thegeneration 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. DISCUSSION 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 42 °Cinmutant 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 42 °C 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 )andtheD 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. 3038 E. S. Bochkareva et al. (Eur. J. Biochem. 269) Ó FEBS 2002 UP12 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 H 2 O 2 or CdCI 2 , 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. coli genes, their promoters are probably recognized by the ÔhousekeepingÕ r 70 factor [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 theUspAfamilyandalsoincomparisonwithallother small cytoplasmic proteins (M r less 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. ACKNOWLEDGEMENTS 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. REFERENCES 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)Roleof 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. Ó FEBS 2002 UP12 is an E. coli universal stress protein (Eur. J. Biochem. 269) 3039 18. 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. 3040 E. S. Bochkareva et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Identification and characterization of the Escherichia coli stress protein UP12, a putative in vivo substrate of GroEL Elena S. Bochkareva, Alexander. 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