Escherichia coli dnaJ deletion mutation results in loss of stability of a positive regulator, CRP.

The dnaJ deletion mutant K7052(lambda dnaK) has a temperature-sensitive defect in the synthesis of beta-galactosidase. We confirmed this operon-specific and temperature-sensitive defect in cell-free extracts prepared from the mutant cells and found that the missing factor was CRP. In the mutant, the cellular concentration of CRP was too low to allow the expression of the lac operon at a nonpermissive temperature. Introduction of a CRP over-producing plasmid into the dnaJ deletion mutant suppressed the defect of beta-galactosidase synthesis. The lower content of CRP in the mutant was found to result from extreme instability of the protein. These results strongly suggested that the heat shock protein dnaJ is involved in the stabilization (or degradation) of CRP.

The dnddeletion mutant K7052(XdnuK) has a temperature-sensitive defect in the synthesis of 0-galactosidase. We confirmed this operon-specific and temperature-sensitive defect in cell-free extracts prepared from the mutant cells and found that the missing factor was CRP. In the mutant, the cellular concentration of CRP was too low to allow the expression of the lac operon at a nonpermissive temperature. Introduction of a CRP over-producing plasmid into the d n d deletion mutant suppressed the defect of &galactosidase synthesis. The lower content of CRP in the mutant was found to result from extreme instability of the protein.
These results strongly suggested that the heat shock protein d n d is involved in the stabilization (or degradation) of CRP.
The dnaJ gene of Escherichia coli was originally found as a cellular gene essential for the multiplication of bacteriophage X (1, 2). Genetic experiments provided evidence for the involvement of the dnaJ protein in the replication of both mini-P and mini-F plasmids (3,4). The dnaJ gene product has also been shown to be essential for cell growth, at least at 43 "C (2,5).
Recently we have reported novel aspects of the phenotype that results from the dnaJ mutation (6). The mutant K7052 in which the whole dnaK-dnaJ operon is deleted (7) and its lysogenic derivative K7052( Xdnak) show a temperature-sensitive defect in the synthesis of @-galactosidase. The synthesis of lac mRNA is reduced at the restrictive temperature (6). The mutants are also conditionally defective in the synthesis of a group of proteins whose synthesis occurred at a specific stage of a cell cycle (8), whereas the synthesis of anthranilate synthetase, encoded by trpED, and other cellular proteins is normal. Lysogenization of the transducing phage containing the wild type dnaJ gene restored the defects (6). From these findings, we proposed that the dnaJ gene product was involved in regulation of the synthesis of these proteins.
In order to elucidate the precise molecular mechanism of the regulation, we attempted to confirm the temperaturesensitive defect of @-galactosidase synthesis in a cell-free protein synthesizing system from the d n a J deletion mutant and to identify the factor that is missing from an extract of the mutant cells grown at 43 "C. In this study, we found that the stability of CRP was decreased in the dnaJ deletion mutant and that the cellular concentration of CRP was lower than the threshold concentration necessary for the expression of the lac operon at a nonpermissive temperature.
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These findings suggested that the heat shock protein, dnaJ, is involved in stabilization or degradation of the positive regulatory protein CRP.

MATERIALS AND METHODS
Bacterial Strains, Bacteriophages, and Plasmids-Bacterial strain K7052 and the transducing phages XdnaK and XdnaKdnaJ have been described (5,9). K7052(Xdnak) was used as a dnaJ deletion mutant (7). K7052(hdnaKdnaJ), which is lysogenized with XdnaKdnaJ instead of with XdnaK, was used as an isogeneic strain carrying the wild type dnaJ gene. lacZ-derivatives of these strains were obtained by nitrosoguanidine mutagenesis and used to prepare cell-free extracts for in uitro @-galactosidase synthesis. The parent lacZ' strain was used in all other experiments throughout this work. CSH73 cells (HfrH, 4Zac A(ara-leu)thi) (10) were used as wild type cells for purification of a complementing factor. Strain CSH44 (F-, tonAA(lac) (Xh80cI857St68) thi(Xh8OcI857St68dlac)) (10) was used for Xh80-dlacDNA preparation. ME5466 (Acrp Acya thi S t f ) (11) was used for plasmid, pKP1013, were kindly donated by Aiba (12) and Miki (131, cloning the crp gene. Plasmids pHA7 and pHA5, and a mini-F respectively. Gene fusion plasmid pTRlO was constructed as follows. A 220base pair AluI-HinfI fragment (nucleotides -40 to +183), which contains the whole control region of the tryptophan operon and the first seven codons of trpE, was isolated from plasmid pMT778 constructed by Hopkins et al. (14). The fragment was inserted at the EcoRI site of plasmid pMC1403 reported by Casadaban et al. (15). In transformed cells, the fusion protein was expressed under the control of the tryptophan promoter; its synthesis was repressed by the presence of tryptophan and induced by the addition of indole acrylate.
Preparation of Cell-free Extracts-Cell-free extracts, S-309, were prepared as described by Zubay (16). Wild type CSH73 cells were grown in nutrient broth (8 g/liter) at 30 "C, while cells of the l a dderivative of the dnaJ deletion mutant, K7052(XdnaK)lacZ were grown in Tris-XY medium (0.1 M Tris-HC1, pH 7.5, 1% tryptone, 0.1% yeast extract, 0.25% NaC1, 20 pg/ml of thymine, and 2 mM CaC12). S-30 extracts prepared from cells grown at 30 "C were named 30"-S-30, and S-30 extracts prepared from cells grown at 30 "C and then further grown at 43 "C for 30 min were named 43"-S-30. I n Vitro Protein Synthesis-Synthesis of @-galactosidase in uitro using an S-30 extract and a template DNA was carried out as described by Zubay (16). Where indicated, in uitro &galactosidase synthesis was separated into two stages, transcription and translation (17).
DNA Preparation-Xh80dlacDNA was prepared from strain CSH44 as described by Zubay (16). Plasmid DNA was prepared described by Maniatis et al. (18).
Enzyme Assay-@-galactosidase was assayed as described previously (19). One unit of enzyme was defined as the amount hydrolyzing 1 pmol of 2-nitrophenyl-@-D-galactopyranoside/h. Binding of [3H] CAMP was measured by the ammonium sulfate precipitation method (20). One unit of CRP was defined as that binding 1 pmol of CAMP.
Electrophoresis and Western Blotting Analysis-SDS'-polyacrylamide gel electrophoresis was carried out as described by Ames (21). electroblotted to nitrocellulose membrane, and the CRP band was Following SDS-polyacrylamide gel electrophoresis, proteins were detected with polyclonal anti-CRP serum, biotinylated rabbit anti-body, streptavidine alkaline phosphatase conjugate, and 5-bromo-4chloro-3-indolyl phosphate with nitro-blue tetrazolium according to the manufacturer's instruction (Amersham). Measurement of the Rate of Synthesis and Stability of CRP-Strains K7052(XdnaK) and K7052(XdnaKdnaJ) were grown to 2 X 10' cells/ ml at 30 "C in Tris-glucose medium containing necessary supplements (6). For measurement of the rate of synthesis of CRP, cultures were divided into two parts. Incubation of one part was continued at 30 "C, while the other part was shifted to 43 "C. After 15 min, the cultures were pulse-laheled with ['4C]leucine (348 mCi/mmol, 50 pCi/ml, Amersham) for 3 min. Samples were then promptly transferred to tubes containing sodium azide (20 mM, final concentration). For measurement of the stability of CRP, cells were labeled with ["C] leucine for 3 min and then chased with excess unlabeled leucine. The cultures were then divided into two parts, which were incubated at 30 and 43 "C, respectively. After various times of chase, 10-ml aliquots were transferred to tubes containing sodium azide. The cells were washed twice with 50 mM Tris-HC1, pH 7.4, containing 0.5 M NaCl and lysed with 1 ml of RIPA buffer (1% deoxycholate, 1% Triton X-100, 0.3 M NaCI, 0.1% SDS, 0.1 M Tris-HCI, pH 7.5, 1 mM phenylmethylsulfonyl fluoride) for 2 h on ice. The supernatants obtained by centrifugation (200,000 X g, 60 min) were supplemented with 20 pl of anti-CRP serum and 5 pg of purified CRP and incubated overnight a t 4 "C. Then 20 pl of protein A (400 mg/ml) was added, and incubation was continued for an additional 2 h. The immunoprecipitates were washed four times with RIPA buffer and boiled in SDS loading buffer for 20 min, and samples were subjected to electrophoresis in 14% SDS-polyacrylamide gel. The amounts of CRP remaining after different chase times were measured with a bioimage analyzer (Fujix BAS 2000).
Cloning of the crp Gene of K7052(XdnaK)-For cloning the crp gene of K7052(XdnaK), genomic libraries were constructed with AD69 as a cloning vector (22). Chromosomal DNA was digested with BamHI and ligated with AD69 phage DNA digested with BamHI. The ligated DNA sample was packaged into phage particles by use of Gigapack gold-10 (Stratagene). The phages were screened by plaque hybridization with a 3.6-kilobase BamHI fragment containing the wild type crp gene isolated from plasmid pHA5 as a probe (12). Recombinant phage DNA containing the crp gene of the mutant was prepared as described by Maniatis et al. (18). The 3.6-kilobase BamHI fragment was isolated and recloned into the unique BamHI site of a single copy plasmid pKP1013 (13), and the recombinant plasmid was named pKP1014. As a control, a 3.6-kilobase BamHI fragment containing the wild type crp gene (obtained from pHA5) was cloned into pKP1013 and the resulting plasmid was named pKP1015.

RESULTS
I n Vitro Synthesis of @-Galactosidase with an Extract of the d n a J Deletion Mutant-As shown in Table I, the S-30 fraction prepared from the lacZderivative of the mutant cells, grown at the permissive temperature (30"-8-30) showed normal ac -TABLE I DNA-directed in vitro synthesis of @-galactosidase with mutant extracts S-30 extracts of K7052 (XdnaK) and CSH73 were prepared as described under "Materials and Methods." Synthetic reactions were run at 37 "C for 90 min. The amount of @-galactosidase synthesized is shown as units/O.l ml of reaction mixture. Amount of protein of cell-free extracts added to 0.1 ml of reaction mixture: K7052 (XdnaK) 30"-S-30, 962 pg; 43"-S-30, 870 pg; wild type S-30, 145 pg. DNA concentrations: Xdlac DNA, 12 pg; pTRlO plasmid DNA, 8 pg. 43"-S-30 -tivity for in vitro @-galactosidase synthesis with XdlacDNA as a template. But after shift of the dnaJ mutant to 43 "C for 30 min, the activity for @-galactosidase synthesis of the resulting cell-free extract (43"-S-30) was 8% of that of 30"-S-30. The addition of a small amount of S-30 from the wild type CSH73 cells to 43"-S-30 restored the activity to almost the same level as that of 30"-S-30.
The plasmid pTRlO contains a trp-lacZ fusion gene in which the promoter region of the lac operon is replaced by that of the tryptophan operon, and a chimeric @-galactosidase can be synthesized under the control of the tryptophan promoter. As shown in Table I, when this plasmid DNA was used as a template, the activity of the 43"-S-30 was 36% of that of 30"-S-30, and addition of a small amount of wild type S-30 did not increase the synthetic activity of 43"-S-30 or 30"-S-30. The reduced synthetic activity of the 43"-S-30 was due to the effect of temperature shift from 30 to 43 "C, not to a defect caused by the dnaJ deletion mutation, because a similar decrease was observed with S-30 prepared from wild type cells subjected to the same temperature shift (data not shown). These results demonstrate that the cell-free extract from the mutant showed a promoter-specific defect in @-galactosidase biosynthesis. An S-30 extract prepared from lacZ' parent strain showed a basal @-galactosidase activity without the addition of template DNA which is due to the @-galactosidase present in the extract. Net in vitro synthesis was calculated by subtracting this value from the total activity observed in the presence of template DNA. We obtained essentially the same result with S-30 prepared from the parent l a d + strain as those shown in Table I.
To determine whether the defect of 43"-S-30 prepared from the mutant is in the transcription or translation step, we separated the overall reaction into transcription and translation steps by synthesizing lac mRNA in the absence of added amino acids and then adding rifampicin and 20 amino acids simultaneously to achieve translation without further initiation of mRNA synthesis. As shown in Table 11, a stimulative effect was observed only when a small amount of the wild type S-30 was added at the step of transcription. These in vitro results are entirely consistent with in vivo results and indicate that transcription of the lacZ gene is blocked at the restrictive temperature.
Identification of the Complementing Factor in the Wild Type S-30 Extract as CRP-During the experiments to purify a complementing factor from S-30 of the wild type cells, we found that the chromatographic behavior of the factor resembled that of CRP, a well-known positive regulator of the lac operon. This finding, together with the observations that both factors function in the transcription step, led us to test the possibility that the two factors might be identical. We meas-TABLE I1 Differential effects of a complementing factor in the process of DNAdirected in vitro @-galactosidase synthesis In vitro @-galactosidase synthesis was separated into transcription and translation steps as described under "Materials and Methods." Transcription was carried out at 30 "C for 7 min, and translation at --ured the cAMP binding activity a t each step of purification and found that cAMP binding activity was copurified with the complementing activity monitored by the ability to restore in vitro @-galactosidase synthesis by 43"-S-30 prepared from the mutant. Moreover, the electrophoretic mobility of the factor on SDS-polyacrylamide gel coincided with that of CRP purified by the method described by Eilen et al. (24), indicating that the complementing factor was CRP.
The cAMP binding activity of 43"-S-30 of the dnaJ deletion mutant was negligible. Analysis of the Bio-Rex 70 column eluate by SDS-polyacrylamide gel electrophoresis also revealed no detectable 22.5-kDa protein band in the fractions in which it was detected in the preparation of wild type cells. Thus, the absence of CRP was responsible for the decrease in the synthesis of @-galactosidase with 43"-S-30 of the dnaJ deletion mutant.
Suppression of the Defect in @-Galactosidase Synthesis by Introduction of a CRP-overproducing Plasmid into the d n a J Deletion Mutant-The plasmid pHA7 contains a 910-base pair fragment which includes the entire structural gene for CRP (12). Strains containing pHA7 overproduce CRP because it is under the control of the bla promoter originally present in pBR322. If the defect of @-galactosidase synthesis in the mutant at the restrictive temperature is due to the absence of CRP, introduction of this plasmid into the mutant should restore the synthesis of @-galactosidase at the restrictive temperature. Fig. 1 shows the time courses of synthesis of @galactosidase in the mutant and the mutant harboring pHA7 at 30 and 43 "C. The rate of synthesis in K7052(XdnaK) was much lower at 43 than at 30 "C ( Fig. lA) as already reported, but similar in the transformant at the two temperatures (Fig.  1B). These findings confirm the conclusion of the in vitro experiments that the defect in the dnaJ deletion mutant in the synthesis of P-galactosidase at 43 "C was due to marked reduction in the cellular concentration of CRP.
Transformation of K7052(XdnaK) with pHA7 did not complement the defect of X phage multiplication a t either temperature or cellular growth at the nonpermissive temperature.
Instability of CRP in the d n a J Deletion Mutant-We have shown that the synthesis of CRP-mRNA in K7052(XdnaK) is almost normal even in cultures a t 43 "C for 40 min (6). This finding suggests that the cellular concentration of CRP is controlled at a post-transcriptional level. Therefore, we compared the rates of synthesis of CRP at 30 and 43 "C in the   iK 0.1 0.2 0.3 0.4 0.2 0.3 0 d n a J mutant. For this, the mutant cells were pulse-labeled with [14C]leucine for 3 min a t 30 or 43 "C, and the labeled proteins were immunoprecipitated with anti-CRP serum. The precipitated proteins were then separated by SDS-polyacrylamide gel electrophoresis. Autoradiograms of the gels showed significant synthesis of CRP at 43 "C, although it was slightly less than at 30 "C (Fig. 2). However, we found that the amount of CRP labeled a t 43 "C relative to that at 30 "C decreased markedly with an increase in the pulse time (data not shown). This finding suggested that the stability of CRP may be lower at the restrictive temperature than at the permissive temperature. The mutant cells were pulse labeled at 30 "C for 3 min with ['4C]leucine and chased at 30 or 43 "C for various times with an excess of unlabeled leucine. The decay rates quantified by autographic analysis of the gel showed that the half-life of CRP was 40 s a t 43 "C and 180 s a t 30 "C (Fig. 3A). Introduction of a transducing phage XdnaKdnuJ into K7052 partially restored the stability of CRP at both temperatures (Fig. 3B), increasing the half-life 2.5-fold (from 40 to 100 s) at the restrictive temperature and 27-fold (from 3 to 80 min) at the permissive temperature. Thus, the lower content of CRP in the dnaJ deletion mutant can mainly be explained by instability of the protein. In the wild type genetic background, no detectable decrease in the amount of CRP was seen during the 60 min chase a t either 30 or 43 "C.
The crp Gene Product of K7052fidnaK) Can Support Expression of the lacZ Gene and Is as Stable as That from the Wild Type Strain ut 43 "C under a dnaJ+ Genetic Back- ground-To exclude the possibility that the crp gene of K7052(hdnaK) was altered during mutagenesis to isolate the mutant and that CRP consequently became unstable at both 30 and 43 "C, we cloned the crp gene of the mutant into a single copy mini-F plasmid pKP1013 and examined whether the crp gene product of the mutant could support @-galactosidase synthesis in a crp deletion mutant, ME5466, at both 30 and 43 "C. Induction of @-galactosidase in ME5466-(pKP1014) carrying the crp gene derived from the dnaJ deletion mutant was almost the same as that in the control transformant ME5466(pKP1015) at both 30 and 43 "C. Furthermore, Western blotting analysis showed that the steady state level of CRP in the pKP1014 transformant grown at 30 "C was the same as that in the pKP1015 transformant. No detectable decrease of CRP content in either transformant was seen after a shift to 43 "C for 30 min (data not shown).
These results indicated that the crp gene product of K7052(XdnaK) has stability similar to that of the wild type strain and that the instability of CRP in K7052(XdnaK) was not due to a mutation of the CRP gene itself.

DISCUSSION
The present study demonstrated that CRP, a well-known positive regulator of catabolite-sensitive operons, was very unstable in the dnddeletion mutant K7052(XdnaK): its halflife in the mutant was 40 s at 43 "C and 180 s at 30 "C ( Fig.  3). The temperature sensitivity of the synthesis of P-galactosidase in the mutant can be explained as follows. The cellular concentration of CRP in the cells grown at 30 "C is lower than that in the wild type cells, but sufficient to support transcription of the lac operon, whereas in cells incubated at 43 "C, its concentration becomes lower than the threshold for supporting expression of the lac operon. The temperaturesensitive defects in the syntheses of other cell cycle-dependent proteins in the mutant (6) can also be explained by CRP instability because almost all the genes for these proteins belong to the catabolite-sensitive operons.
Lysogenization of a transducing phage containing the dnaJ gene derived from wild type cells into the dnaJ mutant increased the stability of CRP 27-fold (from 3 to 80 min) at 30 "C and 2.5-fold (from 40 to 100 s) at 43 "C ( Fig. 3). On the other hand, no detectable degradation of CRP was observed during incubation of wild type cells at 43 "C for 60 min. To exclude the possibility that the amount of dnaJ protein synthesized from XdnaKdnaJ was not sufficient to fully complement the deletion, we compared the cellular levels of dnaJ protein in the wild type parent strain HR9 and K7052-(XdnaKdnaJ). Western blotting analysis shows that the cellular concentration of dnaJ protein in K7052(XdnaKdnaJ) was almost the same as that in HR9 both at 30 and 43 "C (data not shown). The reason that wild type level of stability was not restored in d n d + genetic back ground is not clear. Mutant K7052 was obtained from nitrosoguanidine treated cells by a method designed to select E. coli mutants showing temperature-sensitive growth and a defect in supporting replication of phage X (5). The mutant was considered to have temperature-sensitive defects of both the dnaK and dnaJ genes but recently has been shown to be a deletion mutant lacking the whole dnaK-dnaJ operon (7). Bukau and Walker (4) reported that dnaK deletion mutants were genetically unstable at 30 "C and frequently acquired secondary mutations to grow normally at 30 "C. They showed that the dnuK gene is essential for growth at 30 "C as well as at 43 "C. It is therefore likely that K7052 also has such a suppressor mutation which circumvents the normal requirement of the dnaK gene at 30 "C and signifi-cantly contributes to instability of CRP especially at 43 "C.
Although the lysogenization of XdnaKdnaJ results in the almost complete restoration of @-galactosidase synthesis at 43 "C in vivo, the in vitro synthetic activity of 43"-S-30 prepared from K7052(XdnaKdnaJ) is dependent on the addition of CRP. This can be explained by the fact that CRP is less stable in K7052(XdnuKdnaJ) than in wild type cells, and the concentration of CRP in 43"-S-30 was not sufficient for transcription from the lac promoter.
How do the dnaJ protein and a putative suppressor gene product participate in stabilization or degradation of CRP? A protease responsible for proteolytic degradation of CRP might be induced or activated in the mutant K7052(hdnaK), and this protease might be a heat shock protein since CRP was less stable at 43 than at 30 "C. In this case, the putative degradation system should be specific for CRP or a certain class of proteins since the stabilities of the proteins studied other than CRP were unaffected in the mutant. Another interesting possibility is that the dnaJ protein might interact with CRP and maintain a suitable configuration that protects CRP from the attack by a responsive protease. This possibility is consistent with the idea that heat shock proteins in general act as molecular chaperons to control the higher order conformations of various proteins (25)(26)(27)(28)(29).
There are several reports that heat shock proteins are involved in protein degradation in E. coli. Straus et al. (30) reported that mutations in the heat shock genes, dnaK, d d , grpE, and groEL result in defective proteolysis of abnormal proteins such as puromycyl peptide or a nonsense fragment of p-galactosidase and that overproduction of heat shock proteins increases the rate of decay of the puromycyl fragment. Keller and Simons (23) have also found that a dnaK mutant is defective in the activity to degrade canavanyl proteins and puromycyl peptides but that a temperaturesensitive l a d gene product is degraded more rapidly in the mutant. Studies on the mechanism of the CRP instability in the dnaJ deletion mutant, K7052(XdnaK) should provide new information on heat shock proteins.