JouRNAL OF BACTERIOLOGY, Nov. 1992, p. 7436-7444
Vol.
0021-9193/92/227436-09$02.00/0 Copyright C) 1992, American Society for Microbiology
174, No. 22
Physiological Consequences of DnaK and DnaJ Overproduction in Escherichia coli PAUL BLUM,* JEREMIA ORY, JENNIFER BAUERNFEIND, AND JULIE KRSKA School ofBiological Sciences, University ofNebraska, Lincoln, Nebraska 68588-0118 Received 22 June 1992/Accepted 21 September 1992
The physiological consequences of molecular chaperone overproduction in Escherichia coli are presented. Constitutive overproduction of DnaK from a multicopy plasmid containing large chromosomal fragments spanning the dnaK region resulted in plasmid instability. Co-overproduction of DnaJ with DnaK stabilized plasmid levels. To examine the effects of altered levels of DnaK and DnaJ in a more specific manner, an inducible expression system for dnaK and dnaJ was constructed and characterized. Differential rates of DnaK synthesis were determined by quantitative Western blot (immunoblot) analysis. Moderate levels of DnaK overproduction resulted in a defect in cel septation and formation of cell filaments, but co-overproduction of DnaJ overcame this effect. Further increases in the level of DnaK terminated culture growth despite increased levels of DnaJ. DnaK overproduction was found to be bacteriocidal, and this effect was also partially suppressed by DnaJ. The bacteriocidal effect was apparent only with cultures which were allowed to enter stationary phase, indicating that DnaK toxicity is growth phase dependent.
Molecular chaperones regulate processes involving protein structure. In Escherichia coli, the HSP70 class of chaperones, represented by the heat shock protein DnaK, modulates interactions between proteins involved in the initiation of DNA replication of phage lambda and phage P1 DNA (reviewed in reference 15). DnaK also regulates heat shock protein synthesis (reviewed in reference 16) and contributes to the translocation of some proteins across the cytoplasmic membrane (32). The DnaJ and GrpE proteins (15) have been shown to work together with DnaK in some of these processes. Mutations in the genes for any of these proteins block growth of phage lambda and phage P1, indicating a role for each of them in phage multiplication (2, 14, 34). In vitro analyses have shown that the DnaK protein requires ATP for activity and that its ATPase activity is stimulated by the presence of DnaJ and GrpE (24). The DnaJ protein can play an important role in mediating the specificity of DnaK. DnaJ binds to the phage P1 RepA protein prior to being bound by DnaK in a sequence that results in the release of initiation-competent RepA protein (39). GrpE also binds DnaK (21) and is thought to facilitate the interconversion of DnaK from inactive to active forms (25). Heat shock protein synthesis in E. coli is regulated primarily at the level of transcription initiation (16). Upon a thermal upshift, there is a rapid increase in the transcription rate of heat shock protein genes and therefore in the synthesis of heat shock proteins (40). Heat shock protein synthesis decreases to preshift levels soon after the temperature upshift. Recognition of heat shock promoters is mediated by the alternative sigma factor o-32 (17, 18). &32 concentration plays an important role in setting the level of expression of heat shock genes. c32 levels may be controlled by the levels of DnaK, DnaJ, and GrpE via a homeostatic mechanism involving a2 stability (10) or by a direct effect of temperature on the ATPase activity of DnaK, which in turn could influence a32 levels (26). dnaK null mutations have been constructed, indicating * Corresponding author. Electronic mail address: CRCVMS.UNL.EDU.
PBLUM@ 7436
that DnaK is nonessential, but the physiological consequences of such mutations are severe (30, 31). dnaK mutants are defective in cell septation and are conditionally sensitive for growth above 37°C and below 30°C (7). Such mutants are genetically unstable, and phenotypically normal derivatives occur spontaneously as a result of compensatory mutations in the rpoH gene (8). dnaK mutants are also compromised in their ability to survive during carbon starvation (35) and are sensitive to osmotic stress (27). DnaK is an abundant protein under all conditions and during exponential growth is present at a level of approximately 1% of total protein (36). A 10 to 15°C increase in the temperature of cultivation does not result in significant changes in steady-state levels of DnaK, but prolonged incubation at 46°C increases levels about threefold (36). Little has been reported on the consequences of higher steadystate levels of DnaK on cell physiology, but plasmids which constitutively overproduce DnaK protein have been utilized as a means of enriching cell extracts for the purification of DnaK protein (26, 41) and to control protein aggregation in vivo (5). To understand what the consequences of increased levels of chaperones might be, we systematically examined the effects of DnaK overproduction on the growth, viability, and morphology of E. coli.
MATERIALS AND METHODS Bacterial strains, plasmids, and cell cultivation. Bacterial strains and plasmids used in this study are presented in Table 1. Other plasmids used for plasmid constructions are described in the text. Bacterial strains were cultivated in Luria-Bertani (LB) medium (28). A solid medium was prepared by the addition of 1.5% (wt/vol) Bitek agar (Difco). The medium was supplemented with ampicillin, chloramphenicol, or tetracycline at a concentration of 100, 25, or 10 ,g/ml, respectively, as indicated. Phage P1 vir plaquing and growth of phage lambda were performed as described previously (28). Bacterial inocula for batch culture experiments were prepared by cultivation at 37°C until the cultures had reached mid-log phase; they were then refrigerated until needed. Bacteria were grown at 37°C, and growth was
DnaJ SUPPRESSES DnaK TOXICITY
VOL. 174, 1992
TABLE 1. Bacterial strains and plasmids used in this study Strain or
plasmid0
Strains PBL196 PBL234 PBL325 PBL326 PBL327 PBL328 PBL329 PBL330 GW4813 DH5a
Plasmids pdnaK pMC9 pBN5 pBN6 pBN12 pBN13 pBN15 pBN16 pBN17 pBN18 a
Relevant genotype
Source or reference
pBN5/DH5a pBN6/DH5a recAS6 srl::TnlO lacIq' lacZ::TnS pBN13/PBL325 pBN17/PBL325 pBN15/PBL325 pBN16/PBL325 pBN18/PBL325 dnaK52::Cm F- +80dlacZAMl5 A(lacZYA-argF) U169 recAl end41 hsdR17 (rK-mK+) supE44 thi-1 gyrA relAI
This work This work This work This work This work This work This work This work Graham Walker Laboratory stock
lacFq dnaK+ Cm dnaK+J+ Cm dnaK+J+ bla
Ptac::dnaK+ bla Ptac::dnaK+J+ bla Ptac::dnaJ+ bla Ptac::dnaK+ bla Pt,ac::dnaK A(865-974) bla
Elizabeth Craig Michele Calos 5 This work This work This work This work This work This work This work
All strains listed are E. coli.
monitored spectrophotometrically at a wavelength of 600 nm. Induction of Ptac was accomplished by the addition of isopropyl-o-D-thiogalactopyranoside (IPTG) (Sigma) at the concentrations indicated. Photomicrographs. Cultures were grown and harvested as indicated (see legend to Fig. 3). Heat-fixed cell smears were stained with crystal violet for 1 min, rinsed briefly with water, and then stained with Gram's iodine for 1 min and again briefly rinsed with water. Stained cells were photographed under a bright field. Plasmid constructions. Plasmid isolation and subcloning procedures were performed as described previously (33). pBN5 was constructed by subcloning the 5.3-kb HindIII dnaK fragment from pdnaK (3) into the HindIII site of pACYC184, thereby inactivating the tetracycline resistance gene. pBN6 was constructed by subcloning the 8-kb BamHI fragment from lambda phage 9E4 (22) into the BamHI site of pACYC184 (9). A functional test for DnaK activity was performed with all dnaK-containing plasmids as determined by their ability to complement the inability of the dnaK mutant strain GW4813 to support growth of phage P1. Efficiencies of phage plating for all transformants were found to be the same as those determined for dnaK+ strains. Construction of the inducible P,ac::dnaK+ plasmid, pBN13, was as follows. The 965-bp NruI-PstI dnaK fragment (3) from pBN6 was subcloned into the SmaI-PstI sites of pUC119 (37), creating pBN7. The Hinfl-PstI dnaK fragment (3) from pBN7 was subcloned into the SmaI-PstI sites of pBN8 (see below) by first blunting the ends of fragments derived from a Hinfl partial digest of pBN7, digesting these fragments with PstI, and ligating the resulting DNAs into pBN8 to create pBN9. pBN8 was derived from pKK223-3 (1) but contains a blunted EcoRI laclIq gene fragment from pMC9 (29) cloned into the pKK223-3 NruI site and also lacks the EcoRI site in the pKK223-3 multiple cloning site. The 3'
7437
end of the dnaK gene was then reconstructed by ligation of the EcoRI-HindIII 1.4-kb dnaK fragment from pBN5 into the EcoRI-HindIII site of pBN9 to make pBN13. The dnaK mutant plasmid, pBN18, was constructed by partial digestion of pBN13 with PstI. pBN12 was constructed by subcloning the 8-kb BamHI fragment from pBN6 into the BamHI site of pBR322 (6). The inducible P,ac::dnaK+J+ plasmid, pBN15, was constructed by subcloning a HindIII partial fragment from pBN12 containing the dnaJ region into the HindIII site of pBN13. Insert orientation was determined by the sizes of fragments produced after digestion with BamHI. The dnaJ-deleted derivative of pBN15, called pBN17, was constructed by removal of the dnaJ-containing HindIII fragments. Construction of the inducible P,ac::dnafJ plasmid, pBN16, was accomplished by ligating the 6.53-kb SalI pBN12 fragment containing the dnaJ gene with the 0.5-kb SalI fragment from pKK223-3. The orientation of the Ptac promoter fragment relative to dnaJ was determined by analysis of fragments produced by digestion with BamHI. Plasmid stability experiments. Strains containing plasmids which constitutively express dnaK or dnaKl were first grown with selection for plasmids in medium containing antibiotic. After these cultures had reached stationary phase, cell densities were determined by direct counting in a Petroff-Hausser chamber. The cells were then subcultured 1:1,000 into fresh medium lacking antibiotic and again grown to stationary phase. The number of plasmid-containing cells was determined by plating serial dilutions of these cultures on LB plates with and without antibiotic. The numbers of antibiotic-resistant colonies were normalized against the numbers of colonies which appeared on medium lacking antibiotic and for the total number of cells plated. Only recA mutant strains were used, and all samples were plated in duplicate and the numbers were averaged. Plasmid stability experiments involving plasmids with inducible promoters are described in Results. Protein gel electrophoresis and Western blot (immunoblot) analysis. Proteins were resolved by one-dimensional (1-D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (23) on Bio-Rad minigel rigs by using either prestained or unstained Bio-Rad low-molecular-weight markers. Densitometric analysis of Coomassie blue-stained gels utilized a BioImage densitometer, and analysis of Western blots used a Hoeffer GS300 scanning densitometer. Western blots were prepared as described previously (19). The primary antibody used for probing the Western blots was anti-DnaK mouse polyclonal serum (or preimmune serum), and the secondary antibody was a goat anti-mouse immunoglobulin G alkaline phosphatase conjugate (Sigma). Blots were developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (19). Prior to use in Western blot analysis, the primary antibodies were preadsorbed with an acetone powder prepared with strain GW4813 as described previously (19). Internal DnaK protein standards were included in all Western blots used for quantitative analyses. DnaK values determined by densitometric scanning were normalized against the internal standard prior to interpolation from the standard curve. Protein concentrations were determined by using the bicinchoninic acid assay system (Pierce) and bovine serum albumin protein standards. Preparation of anti-DnaK antibodies. DnaK protein was prepared as follows. Fraction II was prepared as described previously (13, 20) from E. coli PBL234 and fractionated by anion exchange on Mono Q fast protein liquid chromatogra-
7438
J. BA=rRIOL.
BLUM ET AL.
phy. Fractions were examined by 1-D SDS-PAGE, and those containing DnaK protein were pooled, dialyzed into the appropriate loading buffer, and subjected to affinity chromatography on ATP agarose as described previously (38). Selected fractions were pooled and evaluated by 1-D SDS-PAGE. Protein was visualized by Coomassie blue staining (12) and silver staining (12) and later by Western blot (19). The dnaK deletion strain, GW4813 (31), was used as a negative control to facilitate the location of DnaK protein on gels. The ATP-binding fractions containing DnaK were denatured by PAGE, electroeluted, and used to prepare mouse polyclonal antibodies (19). The purity of this material was confirmed by SDS-PAGE and silver staining of both 1and 20-pg quantities of protein. The specificity of the polyclonal antibody sera from five animals was evaluated with purified DnaK protein by 1-D Western blot analysis and by using dnaK+ and dnaK mutant strain extracts. Signals were obtained with all antisera from all mice at 1:1,000 dilutions of both primary and secondary antibodies and with 1- to 10-ng quantities of purified DnaK protein. No signal was obtained with preimmune serum from any of the animals. The results shown here are all for serum (or preimmune serum) from a single mouse. RESULTS Constitutive overproduction of DnaK and DnaJ. To explore the effect of DnaK overproduction on cell physiology, we initially examined plasmids which constitutively expressed dnaK. pBN5 (Table 1) is typical of such plasmids (5, 26, 41) and encodes the dnaK gene under control of its c&2-dependent promoters. A strain (PBL196) containing this plasmid formed mucoid colonies on LB plates at 30°C and to a lesser extent at 37°C. Propagation of PBL196 in liquid culture in the absence of antibiotic selection resulted in rapid loss of plasmid-bearing cells. After 10 generations, only 0.67% of the population retained the plasmid compared with 98% of the population for a control strain which contained the plasmid pACYC184, which lacked the dnaK chromosomal insert. The dnaK and dnaJ genes are cotranscribed in the E. coli chromosome, suggesting that their levels in vivo may be purposely coordinated. We therefore examined the effect of coexpression of the dnaJ and dnaK genes on plasmid stability by using plasmid pBN6. The DNA sequence context of the dnaJ gene relative to the dnaK gene in pBN6 was identical to that present on the E. coli chromosome and utilized the same plasmid replicon, P15A, as pBN5. This ensured that the relative levels of translation of the two genes would remain intact and that the transcription of the genes would continue to initiate from the same cr2-dependent promoters as with pBN5. Strain PBL234, containing pBN6, did not form mucoid colonies on LB plates. In addition, pBN6 was more stable in the absence of antibiotic selection. After 10 generations, 77% of the population retained the plasmid. Overproduction of DnaK with an inducible dnaK expression plasmid. The experiments described above were consistent with the interpretation that increased levels of DnaJ suppressed DnaK plasmid instability, but the plasmids employed in these experiments contained large chromosomal DNA regions flanking the dnaK and dnaJ genes which might also play a role in this process. This possibility was of some concern because of a recent report indicating that a locus that may play a role in some of the phenotypes associated with dnaK mutations exists 5' to the dnaK gene (11). In
FIG. 1. Construction of inducible expression vectors for dnaK,
dnaKJ, and dnaJ.
addition, the plasmids which constitutively overexpress DnaK and DnaJ utilize the natural &2 promoter, leaving open the possibility that alterations in the levels of DnaK and DnaJ could influence their own expression via changes in 9-2 stability, as has been suggested to occur (10). Regulation of expression of dnaK and dnaJ with an artificial inducible promoter system would exclude this possibility. We therefore constructed plasmids lacking all chromosomal DNA 5' to the dnaK gene and in addition replaced the native heat shock promoter with the inducible Ptac promoter system
(Fig. 1).
The effect of dnaK induction on culture growth at a range of IPTG concentrations was determined (Fig. 2). Strain PBL326 contains the plasmid pBN13, which has the dnaK gene under the control of the Ptac promoter. Both the plasmid and the host strain carry the lacI gene (Table 1). Addition of IPTG at concentrations of up to 10 ,M at the time point indicated by the arrow in Fig. 2 did not result in significant growth inhibition, but higher concentrations were severely growth inhibitory. At 10 ,uM IPTG, a protein with an apparent molecular mass of between 70 and 72 kDa increased significantly in abundance as indicated by the upper arrow in Fig. 3A (lanes 1 and 2). The level of this protein as determined by densitometry of Coomassie bluestained gels increased 13.6-fold over that present in uninduced cultures. The identity of this protein was confirmed as DnaK by Western blot analysis with mouse anti-DnaK polyclonal antiserum (Fig. 3B). Quantitative Western blot analysis was used to directly
VOL. 174, 1992
DnaJ SUPPRESSES DnaK TOXICITY 10
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assess levels of DnaK and to explore how the Ptac promoter could be used to regulate expression and overproduction of DnaK. A Western blot of purified DnaK protein ranging in amount from 1 to 256 ng in twofold-increasing increments was probed with anti-DnaK polyclonal antibodies (Fig. 4). The limit of detection was 0.5 ng of DnaK protein. DnaK degradation products were evident at the higher DnaK concentrations examined but were disregarded for the generation of the standard curve. Linear regression analysis of the average peak areas was performed to generate the DnaK standard curve (Fig. 5). The standard curve was used to determine the amounts of DnaK protein present in sample extracts.
Differential rates of synthesis of DnaK protein were determined in strain PBL326 during exponential growth without induction and after induction with 30 and 1,000 ,M IPTG. Cultures were induced and assayed after 10 generations in exponential phase, and DnaK concentrations were determined by Western blot. The amounts of DnaK protein detected were then plotted as a function of increasing cell mass (Fig. 6). The values determined were 0.03 (uninduced), 0.22 (30 FM IPTG), and 0.63 (1,000 ,uM IPTG) ,g of DnaK synthesized per min per ,g of cell protein. The level of DnaK in an otherwise isogenic strain, PBL325 (Table 1), which lacks the dnaK expression plasmid, was 16.1 ng of DnaK per ,g of cell protein, which is equivalent to 1.6% of the total protein. Therefore, uninduced cultures of PBL326, when grown as described in this experiment, have nearly 63% more DnaK protein than that observed for wild-type strains. In addition to the inhibition of growth, DnaK overproduction also led to defective cell septation. In the absence of added IPTG, PBL326 cells were of normal length (Fig. 7A), but the addition of 10 FM IPTG led to formation of elongated rods or filaments many times the normal length (Fig. 7E). At higher IPTG concentrations, filamentous cells were no longer seen; instead, cultures contained cells two to three times the normal length with irregularly swollen regions. On solid media, the addition of IPTG at concentrations that
-27 .5
FIG. 3. Overproduction of DnaK and DnaJ. Strains were grown in LB medium with ampicillin, and IPTG was added at an optical density at 600 nm of 0.1 to a final concentration of 10 PM. Cells were harvested after 2.5 h. Extracts were prepared, and 125 ±g of total protein was loaded per lane on a 12.5% acrylamide gel. (A) Coomassie blue-stained gel. (B) Western blot of a gel identical to that shown in panel A probed with mouse anti-DnaK polyclonal antibodies. The arrow in panel B and the top arrow in panel A indicate the position of DnaK. The lower arrow in panel A indicates the position of DnaJ. Lanes (containing extracts from strains): 1 and 2, PBL326; 3 and 4, PBL328; 5 and 6, PBL327; 7 and 8, PBL329. Lanes 1, 3, 5, and 7 were uninduced, and lanes 2, 4, 6, and 8 were induced with IPTG. MW, molecular weight.
elicited DnaK overproduction and defective cell septation also led to formation of the mucoid colony phenotype. This suggests that DnaK overproduction was the cause of the phenotype in strains with plasmids that constitutively overproduce the protein, such as strain PBL196.
1
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DnaK (ng) 8 16 32
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128 256
FIG. 4. Western blot of a dilution series of purified DnaK protein.
J. BAcTERIOL.
BLUM ET AL.
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DnaK overproduction is bacteriocidal. Growth inhibition and defective cell septation are indicators of abnormal physiology. The consequences of DnaK overproduction for cell viability were therefore examined as an additional indicator of DnaK toxicity. PBL326 was grown to early log phase (A6m = 0.1), and dnaK expression was induced by the addition of IPTG at final concentrations of 1, 3, 10, 30, 100, and 1,000 ,uM. After 3.5 h (stationary phase), samples were removed and the total numbers of cells were determined by direct microscopic examination. Samples were also plated to determine the number of viable cells. Plasmid selection was maintained throughout the experiment. Viable counts were normalized to the total number of cells plated, and the numbers determined were plotted as percentages of the number of viable cells present in uninduced cultures (Fig. 8, closed squares). A progressive decrease in cell viability with increasing concentrations of inducer was seen. The most
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FIG. 7. Effect of DnaK and DnaJ overproduction on cell morphology. Cells were grown in duplicate cultures, and IPTG (10 F.M) was added to one culture of each strain. After 2.5 h, cells were harvested, stained, and photographed (magnification, x400) as described in Materials and Methods. Cultures in panels A, B, C, and D were uninduced, and cultures in panels E, F, G, and H were induced. Strains shown are PBL326 (A and E), PBL328 (B and F), PBL327 (C and G), and PBL329 (D and H). Bars, 5 ,um.
30
significant change in viability occurred at 30 ,M IPTG, where viable cell numbers decreased almost 10,000-fold. At 1,000 p,M IPTG, there was a nearly complete loss of culture viability. The magnitude of the reduction in cell viability following dnaK induction indicated that there might be significant
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Levels of DnaK
determined from cultures of PBL326 growing in exponenitial phase. Samples were removed either immediately before add ition of IPTG or at 15, 30, and 60 min after addition of IPTG. Sample concentrations: no IPTG (crosses); 30 ILM IPTG (closed triangles). circles); 1,000 puM IPTG (closed I
protein
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DnaK. All colonies which appeared on plates containing 1,000 puM IPTG (70 colonies tested) had lost the IPTGsensitive phenotype, whereas about 60% of cells from plates containing 30 p,M IPTG (84 colonies tested) retained the wild-type phenotype and 100% of cells from plates containing 0, 1, 3, and 10 pM IPTG (100 colonies tested) retained the wild-type phenotype. These results indicated that pBN13 would be preferentially lost under inducing conditions in the absence of
VOL. 174, 1992
DnaJ SUPPRESSES DnaK TOXICITY
100
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IPTG (gM) FIG. 8. Bacteriocidal activity of DnaK overproduction. Cultures of PBL326 (closed squares) and PBL328 (closed circles) in exponential phase were treated with IPTG at the concentrations indicated and were then allowed to enter stationary phase. Samples were removed, and total numbers and viable cell numbers were determined.
antibiotic selection. The stability of this plasmid was therefore evaluated after induction by treatment with 30 FM IPTG. PBL326 was grown to mid-log phase (A6w = 0.2) in LB ampicillin medium and subcultured at a dilution of 256-fold into two flasks, one of which contained 30 ,uM IPTG. The cultures were sampled after 10 generations in exponential phase (A6. = 0.8) and after entry into stationary phase (A600 = 4.0). Diluted samples were plated on LB plates with and without ampicillin and incubated to determine the percentage of plasmid-bearing cells (Table 2). The presence of IPTG in the culture had no significant effect on plasmid retention in samples derived from exponential-phase cultures. However, when cultures were allowed to reach stationary phase, the percentage of plasmid-bearing cells in IPTG-treated culture was reduced greatly. Fewer than 1 in 1,000 colonies which arose on LB ampicillin plates retained the IPTG-sensitive phenotype; all colonies tested appeared to have lost the wild-type IPTG-sensitive phenotype. This result indicates that the toxicity of DnaK overproduction is growth phase dependent and that cultures in exponential phase can tolerate relatively high levels of DnaK protein without loss of viability. Entry into stationary phase appears to be lethal even for cells which are overproducing this protein. The extent of mutant overgrowth of the induced TABLE 2. Plasmid stability in PBL326 and PBL328 treated with 30 pM IPTG"
Strain
PBL326
Addition of: IPTG Ampicillin
+
+ + -
+
+
+
+ -
No. of viable cells/ml of culture Exponential phase Stationary phase 2.5 x 108 3.8 x 107 6.9 x 107
Vol.
0021-9193/92/227436-09$02.00/0 Copyright C) 1992, American Society for Microbiology
174, No. 22
Physiological Consequences of DnaK and DnaJ Overproduction in Escherichia coli PAUL BLUM,* JEREMIA ORY, JENNIFER BAUERNFEIND, AND JULIE KRSKA School ofBiological Sciences, University ofNebraska, Lincoln, Nebraska 68588-0118 Received 22 June 1992/Accepted 21 September 1992
The physiological consequences of molecular chaperone overproduction in Escherichia coli are presented. Constitutive overproduction of DnaK from a multicopy plasmid containing large chromosomal fragments spanning the dnaK region resulted in plasmid instability. Co-overproduction of DnaJ with DnaK stabilized plasmid levels. To examine the effects of altered levels of DnaK and DnaJ in a more specific manner, an inducible expression system for dnaK and dnaJ was constructed and characterized. Differential rates of DnaK synthesis were determined by quantitative Western blot (immunoblot) analysis. Moderate levels of DnaK overproduction resulted in a defect in cel septation and formation of cell filaments, but co-overproduction of DnaJ overcame this effect. Further increases in the level of DnaK terminated culture growth despite increased levels of DnaJ. DnaK overproduction was found to be bacteriocidal, and this effect was also partially suppressed by DnaJ. The bacteriocidal effect was apparent only with cultures which were allowed to enter stationary phase, indicating that DnaK toxicity is growth phase dependent.
Molecular chaperones regulate processes involving protein structure. In Escherichia coli, the HSP70 class of chaperones, represented by the heat shock protein DnaK, modulates interactions between proteins involved in the initiation of DNA replication of phage lambda and phage P1 DNA (reviewed in reference 15). DnaK also regulates heat shock protein synthesis (reviewed in reference 16) and contributes to the translocation of some proteins across the cytoplasmic membrane (32). The DnaJ and GrpE proteins (15) have been shown to work together with DnaK in some of these processes. Mutations in the genes for any of these proteins block growth of phage lambda and phage P1, indicating a role for each of them in phage multiplication (2, 14, 34). In vitro analyses have shown that the DnaK protein requires ATP for activity and that its ATPase activity is stimulated by the presence of DnaJ and GrpE (24). The DnaJ protein can play an important role in mediating the specificity of DnaK. DnaJ binds to the phage P1 RepA protein prior to being bound by DnaK in a sequence that results in the release of initiation-competent RepA protein (39). GrpE also binds DnaK (21) and is thought to facilitate the interconversion of DnaK from inactive to active forms (25). Heat shock protein synthesis in E. coli is regulated primarily at the level of transcription initiation (16). Upon a thermal upshift, there is a rapid increase in the transcription rate of heat shock protein genes and therefore in the synthesis of heat shock proteins (40). Heat shock protein synthesis decreases to preshift levels soon after the temperature upshift. Recognition of heat shock promoters is mediated by the alternative sigma factor o-32 (17, 18). &32 concentration plays an important role in setting the level of expression of heat shock genes. c32 levels may be controlled by the levels of DnaK, DnaJ, and GrpE via a homeostatic mechanism involving a2 stability (10) or by a direct effect of temperature on the ATPase activity of DnaK, which in turn could influence a32 levels (26). dnaK null mutations have been constructed, indicating * Corresponding author. Electronic mail address: CRCVMS.UNL.EDU.
PBLUM@ 7436
that DnaK is nonessential, but the physiological consequences of such mutations are severe (30, 31). dnaK mutants are defective in cell septation and are conditionally sensitive for growth above 37°C and below 30°C (7). Such mutants are genetically unstable, and phenotypically normal derivatives occur spontaneously as a result of compensatory mutations in the rpoH gene (8). dnaK mutants are also compromised in their ability to survive during carbon starvation (35) and are sensitive to osmotic stress (27). DnaK is an abundant protein under all conditions and during exponential growth is present at a level of approximately 1% of total protein (36). A 10 to 15°C increase in the temperature of cultivation does not result in significant changes in steady-state levels of DnaK, but prolonged incubation at 46°C increases levels about threefold (36). Little has been reported on the consequences of higher steadystate levels of DnaK on cell physiology, but plasmids which constitutively overproduce DnaK protein have been utilized as a means of enriching cell extracts for the purification of DnaK protein (26, 41) and to control protein aggregation in vivo (5). To understand what the consequences of increased levels of chaperones might be, we systematically examined the effects of DnaK overproduction on the growth, viability, and morphology of E. coli.
MATERIALS AND METHODS Bacterial strains, plasmids, and cell cultivation. Bacterial strains and plasmids used in this study are presented in Table 1. Other plasmids used for plasmid constructions are described in the text. Bacterial strains were cultivated in Luria-Bertani (LB) medium (28). A solid medium was prepared by the addition of 1.5% (wt/vol) Bitek agar (Difco). The medium was supplemented with ampicillin, chloramphenicol, or tetracycline at a concentration of 100, 25, or 10 ,g/ml, respectively, as indicated. Phage P1 vir plaquing and growth of phage lambda were performed as described previously (28). Bacterial inocula for batch culture experiments were prepared by cultivation at 37°C until the cultures had reached mid-log phase; they were then refrigerated until needed. Bacteria were grown at 37°C, and growth was
DnaJ SUPPRESSES DnaK TOXICITY
VOL. 174, 1992
TABLE 1. Bacterial strains and plasmids used in this study Strain or
plasmid0
Strains PBL196 PBL234 PBL325 PBL326 PBL327 PBL328 PBL329 PBL330 GW4813 DH5a
Plasmids pdnaK pMC9 pBN5 pBN6 pBN12 pBN13 pBN15 pBN16 pBN17 pBN18 a
Relevant genotype
Source or reference
pBN5/DH5a pBN6/DH5a recAS6 srl::TnlO lacIq' lacZ::TnS pBN13/PBL325 pBN17/PBL325 pBN15/PBL325 pBN16/PBL325 pBN18/PBL325 dnaK52::Cm F- +80dlacZAMl5 A(lacZYA-argF) U169 recAl end41 hsdR17 (rK-mK+) supE44 thi-1 gyrA relAI
This work This work This work This work This work This work This work This work Graham Walker Laboratory stock
lacFq dnaK+ Cm dnaK+J+ Cm dnaK+J+ bla
Ptac::dnaK+ bla Ptac::dnaK+J+ bla Ptac::dnaJ+ bla Ptac::dnaK+ bla Pt,ac::dnaK A(865-974) bla
Elizabeth Craig Michele Calos 5 This work This work This work This work This work This work This work
All strains listed are E. coli.
monitored spectrophotometrically at a wavelength of 600 nm. Induction of Ptac was accomplished by the addition of isopropyl-o-D-thiogalactopyranoside (IPTG) (Sigma) at the concentrations indicated. Photomicrographs. Cultures were grown and harvested as indicated (see legend to Fig. 3). Heat-fixed cell smears were stained with crystal violet for 1 min, rinsed briefly with water, and then stained with Gram's iodine for 1 min and again briefly rinsed with water. Stained cells were photographed under a bright field. Plasmid constructions. Plasmid isolation and subcloning procedures were performed as described previously (33). pBN5 was constructed by subcloning the 5.3-kb HindIII dnaK fragment from pdnaK (3) into the HindIII site of pACYC184, thereby inactivating the tetracycline resistance gene. pBN6 was constructed by subcloning the 8-kb BamHI fragment from lambda phage 9E4 (22) into the BamHI site of pACYC184 (9). A functional test for DnaK activity was performed with all dnaK-containing plasmids as determined by their ability to complement the inability of the dnaK mutant strain GW4813 to support growth of phage P1. Efficiencies of phage plating for all transformants were found to be the same as those determined for dnaK+ strains. Construction of the inducible P,ac::dnaK+ plasmid, pBN13, was as follows. The 965-bp NruI-PstI dnaK fragment (3) from pBN6 was subcloned into the SmaI-PstI sites of pUC119 (37), creating pBN7. The Hinfl-PstI dnaK fragment (3) from pBN7 was subcloned into the SmaI-PstI sites of pBN8 (see below) by first blunting the ends of fragments derived from a Hinfl partial digest of pBN7, digesting these fragments with PstI, and ligating the resulting DNAs into pBN8 to create pBN9. pBN8 was derived from pKK223-3 (1) but contains a blunted EcoRI laclIq gene fragment from pMC9 (29) cloned into the pKK223-3 NruI site and also lacks the EcoRI site in the pKK223-3 multiple cloning site. The 3'
7437
end of the dnaK gene was then reconstructed by ligation of the EcoRI-HindIII 1.4-kb dnaK fragment from pBN5 into the EcoRI-HindIII site of pBN9 to make pBN13. The dnaK mutant plasmid, pBN18, was constructed by partial digestion of pBN13 with PstI. pBN12 was constructed by subcloning the 8-kb BamHI fragment from pBN6 into the BamHI site of pBR322 (6). The inducible P,ac::dnaK+J+ plasmid, pBN15, was constructed by subcloning a HindIII partial fragment from pBN12 containing the dnaJ region into the HindIII site of pBN13. Insert orientation was determined by the sizes of fragments produced after digestion with BamHI. The dnaJ-deleted derivative of pBN15, called pBN17, was constructed by removal of the dnaJ-containing HindIII fragments. Construction of the inducible P,ac::dnafJ plasmid, pBN16, was accomplished by ligating the 6.53-kb SalI pBN12 fragment containing the dnaJ gene with the 0.5-kb SalI fragment from pKK223-3. The orientation of the Ptac promoter fragment relative to dnaJ was determined by analysis of fragments produced by digestion with BamHI. Plasmid stability experiments. Strains containing plasmids which constitutively express dnaK or dnaKl were first grown with selection for plasmids in medium containing antibiotic. After these cultures had reached stationary phase, cell densities were determined by direct counting in a Petroff-Hausser chamber. The cells were then subcultured 1:1,000 into fresh medium lacking antibiotic and again grown to stationary phase. The number of plasmid-containing cells was determined by plating serial dilutions of these cultures on LB plates with and without antibiotic. The numbers of antibiotic-resistant colonies were normalized against the numbers of colonies which appeared on medium lacking antibiotic and for the total number of cells plated. Only recA mutant strains were used, and all samples were plated in duplicate and the numbers were averaged. Plasmid stability experiments involving plasmids with inducible promoters are described in Results. Protein gel electrophoresis and Western blot (immunoblot) analysis. Proteins were resolved by one-dimensional (1-D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (23) on Bio-Rad minigel rigs by using either prestained or unstained Bio-Rad low-molecular-weight markers. Densitometric analysis of Coomassie blue-stained gels utilized a BioImage densitometer, and analysis of Western blots used a Hoeffer GS300 scanning densitometer. Western blots were prepared as described previously (19). The primary antibody used for probing the Western blots was anti-DnaK mouse polyclonal serum (or preimmune serum), and the secondary antibody was a goat anti-mouse immunoglobulin G alkaline phosphatase conjugate (Sigma). Blots were developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (19). Prior to use in Western blot analysis, the primary antibodies were preadsorbed with an acetone powder prepared with strain GW4813 as described previously (19). Internal DnaK protein standards were included in all Western blots used for quantitative analyses. DnaK values determined by densitometric scanning were normalized against the internal standard prior to interpolation from the standard curve. Protein concentrations were determined by using the bicinchoninic acid assay system (Pierce) and bovine serum albumin protein standards. Preparation of anti-DnaK antibodies. DnaK protein was prepared as follows. Fraction II was prepared as described previously (13, 20) from E. coli PBL234 and fractionated by anion exchange on Mono Q fast protein liquid chromatogra-
7438
J. BA=rRIOL.
BLUM ET AL.
phy. Fractions were examined by 1-D SDS-PAGE, and those containing DnaK protein were pooled, dialyzed into the appropriate loading buffer, and subjected to affinity chromatography on ATP agarose as described previously (38). Selected fractions were pooled and evaluated by 1-D SDS-PAGE. Protein was visualized by Coomassie blue staining (12) and silver staining (12) and later by Western blot (19). The dnaK deletion strain, GW4813 (31), was used as a negative control to facilitate the location of DnaK protein on gels. The ATP-binding fractions containing DnaK were denatured by PAGE, electroeluted, and used to prepare mouse polyclonal antibodies (19). The purity of this material was confirmed by SDS-PAGE and silver staining of both 1and 20-pg quantities of protein. The specificity of the polyclonal antibody sera from five animals was evaluated with purified DnaK protein by 1-D Western blot analysis and by using dnaK+ and dnaK mutant strain extracts. Signals were obtained with all antisera from all mice at 1:1,000 dilutions of both primary and secondary antibodies and with 1- to 10-ng quantities of purified DnaK protein. No signal was obtained with preimmune serum from any of the animals. The results shown here are all for serum (or preimmune serum) from a single mouse. RESULTS Constitutive overproduction of DnaK and DnaJ. To explore the effect of DnaK overproduction on cell physiology, we initially examined plasmids which constitutively expressed dnaK. pBN5 (Table 1) is typical of such plasmids (5, 26, 41) and encodes the dnaK gene under control of its c&2-dependent promoters. A strain (PBL196) containing this plasmid formed mucoid colonies on LB plates at 30°C and to a lesser extent at 37°C. Propagation of PBL196 in liquid culture in the absence of antibiotic selection resulted in rapid loss of plasmid-bearing cells. After 10 generations, only 0.67% of the population retained the plasmid compared with 98% of the population for a control strain which contained the plasmid pACYC184, which lacked the dnaK chromosomal insert. The dnaK and dnaJ genes are cotranscribed in the E. coli chromosome, suggesting that their levels in vivo may be purposely coordinated. We therefore examined the effect of coexpression of the dnaJ and dnaK genes on plasmid stability by using plasmid pBN6. The DNA sequence context of the dnaJ gene relative to the dnaK gene in pBN6 was identical to that present on the E. coli chromosome and utilized the same plasmid replicon, P15A, as pBN5. This ensured that the relative levels of translation of the two genes would remain intact and that the transcription of the genes would continue to initiate from the same cr2-dependent promoters as with pBN5. Strain PBL234, containing pBN6, did not form mucoid colonies on LB plates. In addition, pBN6 was more stable in the absence of antibiotic selection. After 10 generations, 77% of the population retained the plasmid. Overproduction of DnaK with an inducible dnaK expression plasmid. The experiments described above were consistent with the interpretation that increased levels of DnaJ suppressed DnaK plasmid instability, but the plasmids employed in these experiments contained large chromosomal DNA regions flanking the dnaK and dnaJ genes which might also play a role in this process. This possibility was of some concern because of a recent report indicating that a locus that may play a role in some of the phenotypes associated with dnaK mutations exists 5' to the dnaK gene (11). In
FIG. 1. Construction of inducible expression vectors for dnaK,
dnaKJ, and dnaJ.
addition, the plasmids which constitutively overexpress DnaK and DnaJ utilize the natural &2 promoter, leaving open the possibility that alterations in the levels of DnaK and DnaJ could influence their own expression via changes in 9-2 stability, as has been suggested to occur (10). Regulation of expression of dnaK and dnaJ with an artificial inducible promoter system would exclude this possibility. We therefore constructed plasmids lacking all chromosomal DNA 5' to the dnaK gene and in addition replaced the native heat shock promoter with the inducible Ptac promoter system
(Fig. 1).
The effect of dnaK induction on culture growth at a range of IPTG concentrations was determined (Fig. 2). Strain PBL326 contains the plasmid pBN13, which has the dnaK gene under the control of the Ptac promoter. Both the plasmid and the host strain carry the lacI gene (Table 1). Addition of IPTG at concentrations of up to 10 ,M at the time point indicated by the arrow in Fig. 2 did not result in significant growth inhibition, but higher concentrations were severely growth inhibitory. At 10 ,uM IPTG, a protein with an apparent molecular mass of between 70 and 72 kDa increased significantly in abundance as indicated by the upper arrow in Fig. 3A (lanes 1 and 2). The level of this protein as determined by densitometry of Coomassie bluestained gels increased 13.6-fold over that present in uninduced cultures. The identity of this protein was confirmed as DnaK by Western blot analysis with mouse anti-DnaK polyclonal antiserum (Fig. 3B). Quantitative Western blot analysis was used to directly
VOL. 174, 1992
DnaJ SUPPRESSES DnaK TOXICITY 10
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assess levels of DnaK and to explore how the Ptac promoter could be used to regulate expression and overproduction of DnaK. A Western blot of purified DnaK protein ranging in amount from 1 to 256 ng in twofold-increasing increments was probed with anti-DnaK polyclonal antibodies (Fig. 4). The limit of detection was 0.5 ng of DnaK protein. DnaK degradation products were evident at the higher DnaK concentrations examined but were disregarded for the generation of the standard curve. Linear regression analysis of the average peak areas was performed to generate the DnaK standard curve (Fig. 5). The standard curve was used to determine the amounts of DnaK protein present in sample extracts.
Differential rates of synthesis of DnaK protein were determined in strain PBL326 during exponential growth without induction and after induction with 30 and 1,000 ,M IPTG. Cultures were induced and assayed after 10 generations in exponential phase, and DnaK concentrations were determined by Western blot. The amounts of DnaK protein detected were then plotted as a function of increasing cell mass (Fig. 6). The values determined were 0.03 (uninduced), 0.22 (30 FM IPTG), and 0.63 (1,000 ,uM IPTG) ,g of DnaK synthesized per min per ,g of cell protein. The level of DnaK in an otherwise isogenic strain, PBL325 (Table 1), which lacks the dnaK expression plasmid, was 16.1 ng of DnaK per ,g of cell protein, which is equivalent to 1.6% of the total protein. Therefore, uninduced cultures of PBL326, when grown as described in this experiment, have nearly 63% more DnaK protein than that observed for wild-type strains. In addition to the inhibition of growth, DnaK overproduction also led to defective cell septation. In the absence of added IPTG, PBL326 cells were of normal length (Fig. 7A), but the addition of 10 FM IPTG led to formation of elongated rods or filaments many times the normal length (Fig. 7E). At higher IPTG concentrations, filamentous cells were no longer seen; instead, cultures contained cells two to three times the normal length with irregularly swollen regions. On solid media, the addition of IPTG at concentrations that
-27 .5
FIG. 3. Overproduction of DnaK and DnaJ. Strains were grown in LB medium with ampicillin, and IPTG was added at an optical density at 600 nm of 0.1 to a final concentration of 10 PM. Cells were harvested after 2.5 h. Extracts were prepared, and 125 ±g of total protein was loaded per lane on a 12.5% acrylamide gel. (A) Coomassie blue-stained gel. (B) Western blot of a gel identical to that shown in panel A probed with mouse anti-DnaK polyclonal antibodies. The arrow in panel B and the top arrow in panel A indicate the position of DnaK. The lower arrow in panel A indicates the position of DnaJ. Lanes (containing extracts from strains): 1 and 2, PBL326; 3 and 4, PBL328; 5 and 6, PBL327; 7 and 8, PBL329. Lanes 1, 3, 5, and 7 were uninduced, and lanes 2, 4, 6, and 8 were induced with IPTG. MW, molecular weight.
elicited DnaK overproduction and defective cell septation also led to formation of the mucoid colony phenotype. This suggests that DnaK overproduction was the cause of the phenotype in strains with plasmids that constitutively overproduce the protein, such as strain PBL196.
1
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DnaK (ng) 8 16 32
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FIG. 4. Western blot of a dilution series of purified DnaK protein.
J. BAcTERIOL.
BLUM ET AL.
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DnaK overproduction is bacteriocidal. Growth inhibition and defective cell septation are indicators of abnormal physiology. The consequences of DnaK overproduction for cell viability were therefore examined as an additional indicator of DnaK toxicity. PBL326 was grown to early log phase (A6m = 0.1), and dnaK expression was induced by the addition of IPTG at final concentrations of 1, 3, 10, 30, 100, and 1,000 ,uM. After 3.5 h (stationary phase), samples were removed and the total numbers of cells were determined by direct microscopic examination. Samples were also plated to determine the number of viable cells. Plasmid selection was maintained throughout the experiment. Viable counts were normalized to the total number of cells plated, and the numbers determined were plotted as percentages of the number of viable cells present in uninduced cultures (Fig. 8, closed squares). A progressive decrease in cell viability with increasing concentrations of inducer was seen. The most
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FIG. 7. Effect of DnaK and DnaJ overproduction on cell morphology. Cells were grown in duplicate cultures, and IPTG (10 F.M) was added to one culture of each strain. After 2.5 h, cells were harvested, stained, and photographed (magnification, x400) as described in Materials and Methods. Cultures in panels A, B, C, and D were uninduced, and cultures in panels E, F, G, and H were induced. Strains shown are PBL326 (A and E), PBL328 (B and F), PBL327 (C and G), and PBL329 (D and H). Bars, 5 ,um.
30
significant change in viability occurred at 30 ,M IPTG, where viable cell numbers decreased almost 10,000-fold. At 1,000 p,M IPTG, there was a nearly complete loss of culture viability. The magnitude of the reduction in cell viability following dnaK induction indicated that there might be significant
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6. Differential rates of DnaK
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Levels of DnaK
determined from cultures of PBL326 growing in exponenitial phase. Samples were removed either immediately before add ition of IPTG or at 15, 30, and 60 min after addition of IPTG. Sample concentrations: no IPTG (crosses); 30 ILM IPTG (closed triangles). circles); 1,000 puM IPTG (closed I
protein
no
longer overproduce
DnaK protein. The ability of surviving cells to induce DnaK overproduction was therefore evaluated by streaking surviving colonies on LB ampicillin plates with and without 1,000 p-M IPTG. PBL326 is unable to grow on plates containing
were
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DnaK. All colonies which appeared on plates containing 1,000 puM IPTG (70 colonies tested) had lost the IPTGsensitive phenotype, whereas about 60% of cells from plates containing 30 p,M IPTG (84 colonies tested) retained the wild-type phenotype and 100% of cells from plates containing 0, 1, 3, and 10 pM IPTG (100 colonies tested) retained the wild-type phenotype. These results indicated that pBN13 would be preferentially lost under inducing conditions in the absence of
VOL. 174, 1992
DnaJ SUPPRESSES DnaK TOXICITY
100
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IPTG (gM) FIG. 8. Bacteriocidal activity of DnaK overproduction. Cultures of PBL326 (closed squares) and PBL328 (closed circles) in exponential phase were treated with IPTG at the concentrations indicated and were then allowed to enter stationary phase. Samples were removed, and total numbers and viable cell numbers were determined.
antibiotic selection. The stability of this plasmid was therefore evaluated after induction by treatment with 30 FM IPTG. PBL326 was grown to mid-log phase (A6w = 0.2) in LB ampicillin medium and subcultured at a dilution of 256-fold into two flasks, one of which contained 30 ,uM IPTG. The cultures were sampled after 10 generations in exponential phase (A6. = 0.8) and after entry into stationary phase (A600 = 4.0). Diluted samples were plated on LB plates with and without ampicillin and incubated to determine the percentage of plasmid-bearing cells (Table 2). The presence of IPTG in the culture had no significant effect on plasmid retention in samples derived from exponential-phase cultures. However, when cultures were allowed to reach stationary phase, the percentage of plasmid-bearing cells in IPTG-treated culture was reduced greatly. Fewer than 1 in 1,000 colonies which arose on LB ampicillin plates retained the IPTG-sensitive phenotype; all colonies tested appeared to have lost the wild-type IPTG-sensitive phenotype. This result indicates that the toxicity of DnaK overproduction is growth phase dependent and that cultures in exponential phase can tolerate relatively high levels of DnaK protein without loss of viability. Entry into stationary phase appears to be lethal even for cells which are overproducing this protein. The extent of mutant overgrowth of the induced TABLE 2. Plasmid stability in PBL326 and PBL328 treated with 30 pM IPTG"
Strain
PBL326
Addition of: IPTG Ampicillin
+
+ + -
+
+
+
+ -
No. of viable cells/ml of culture Exponential phase Stationary phase 2.5 x 108 3.8 x 107 6.9 x 107
