Expression of the Caulobacter heat shock gene dnaK is developmentally controlled during growth at normal temperatures

Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions.

The heat shock response is an apparently universal phenomenon by which a cell subjected to a sudden increase in temperature induces the synthesis of a small number of highly conserved proteins, the heat shock proteins (hsps) (20,21,26). The high degree of conservation of the hsps suggests that these proteins are involved in important processes within the cell.
The best studied among these proteins is the family of polypeptides of Mr 70,000. Eucaryotic organisms typically contain several hsp70-related genes (17,25,34). Regulation of these genes is complex; some hsp70 genes are expressed only after cells are stressed, whereas others are expressed constitutively or are developmentally regulated. In contrast, Escherichia coli encodes only one hsp70 protein, the product of the dnaK gene (4). DnaK is an abundant protein even at normal temperatures (reported to compose 1.4% of the cellular protein), and its synthesis is induced by heat and other forms of stress (15). It is required for the initiation of lambda phage DNA replication (19), is essential for cellular growth, and appears to be required for the initiation of DNA replication at high temperatures (28,31). We have previously shown that the DnaK homolog from Caulobacter crescentus is under complex regulation. In addition to increased synthesis after brief exposure to heat (13,29) or peroxide (29), under physiologic growth conditions DnaK appears to be synthesized at a defined time in the cell cycle, just before the initiation of DNA replication (13). This response is similar to that observed with the human hsp70 protein, which has been shown in HeLa cells to rapidly increase synthesis during entry into the S phase and to decline in the late-S and G2 phases (23). Furthermore, the human hsp70 protein is local-* Corresponding author.
ized to the nucleus in the S phase. At each cell division in C. crescentus, only the progeny stalked cell is able to initiate DNA replication (8), and DnaK synthesized in the predivisional cell is selectively localized to this cell upon division (29).
To determine whether a single dnaK gene is responsible for both the stress response and the response to temporal control signals, we cloned dnaK; it occurred in a single copy on the genome, and we identified both a conserved heat shock promoter and a conserved Eu70 promoter in tandem 5' to the start of the dnaK coding sequence. Analysis of dnaK transcription in vivo showed that the gene is only transcribed from the heat shock promoter at high temperatures but is transcribed from both promoters in a temporal manner at normal growth temperatures.

MATERIALS AND METHODS
Bacterial strains and plasmids. The synchronizable widetype strain C. crescentus CB15N, used in all experiments, was grown in PYE medium or in modified minimal M2 glucose medium (6). Cultures were synchronized by the method of Evinger and Agabian (12). E. coli HB101 was grown in LB medium (24). Plasmid B8, containing the Drosophila hsp70 gene cloned into pBR322, was obtained from E. Craig. Plasmid pUCD615, containing the promoterless luxCDABE cassette, was obtained from C. I. Kado (16). Plasmid pRK290.20R, a derivative of the broad-host-range plasmid pRK290 (9), contains a multiple restriction site cassette inserted in the EcoRI site.
Cloning of the dnaK gene. C Denhardt solution-0.1% sodium do( washes in 5 x SSC at 37°C) to the Dro the B8 plasmid (18). A single band ( region corresponding to 3.5 kilobases crescentus genomic DNA was then and size fractionated by agarose ge DNA fragments from 2 to 4 kb in size ed and ligated to BamHI-digested pB] genomic library. After transformation ing partial library was screened by prn digests of miniplasmid preparations tion with a nick-translated Drosophilc plasmid. Both methods yielded the fragment. DNA sequencing. DNA sequence 3.5-kb cloned fragment was done by t] et al. (32). Various restriction fragmer M13mpl8 and M13mpl9, and their co determined on both strands after de prepared by using the cyclone syst Biotechnologies. A restriction map o Fig. 1, and the sequence is shown in Nuclease S1 and primer extension n S1 mapping experiments was a 1.1-kb isolated from pGR24 ( Fig. 1). S1 performed as described by Amemi following modifications. Hybridizatic 90°C for 10 min, followed by transfer ,ug of C. crescentus total RNA. Nuc performed with 100 U per sample at 2 extension mapping (3) was done with otide (24-mer) that was complementa 456 of the dnaK coding sequence (Fi tide was obtained from Operon TI Calif. The oligonucleotide was 5' end hybridized to 50 ,ug of C. crescentus was carried out in 25 p1l of 0.1 M ethanesulfonic acid) (pH 6 (2). Luciferase activity was , the restriction map. An assayed in C. crescentus cultures containing plasmid-borne r S1 nuclease protection lux genes. To assay the activity of luciferase at defined time s: B, BamHI; C, ClaI; P, points in the cell cycle, synchronized cultures were incubated at 32°C, and then samples were shifted to 25°C for 10 min at the times indicated in Fig. 6B. Because luciferase is decyl sulfate at 55°C; irreversibly denatured at temperatures greater than 30°C, sophila hsp70 probe in this method provides a means of assaying the new synthesis of hybridization in the of luciferase over a short period of time. After incubation at (kb) was observed. C. 25°C for 10 min, each sample of the culture (optical density digested with BamHI at 660 nm, 0.3 to 1.0) was placed in a scintillation vial, and 1 electrophoresis. The n-decylaldehyde (2% emulsion) was added to a final concenwere then electroelut-tration of 0.002%. Emitted light was measured immediately R322 to create a partial over a period of 6 s in a Packard scintillation counter (model iinto E. coli, the result-3330). Counts obtained on the 3H channel were converted )bing Southern blots of into quanta emitted by using a known radioactive standard )r by colony hybridiza-for calibration (33). 7 hsp70 probe in the B8 same 3.5-kb BamHI RESULTS analysis of the entire Isolation and sequence analysis of the dnaK gene from C. he procedure of Sanger crescentus. A single segment of DNA in the C. crescentus its were subcloned into genome was identified by hybridization of the Drosophila mplete sequences were hsp70 probe B8 (18) to genomic DNA blots. DNA isolated letion subclones were from the region of the gel that hybridized to the probe was :em from International used to construct a BamHI plasmid library in pBR322, as if the clone is shown in described in Materials and Methods. Two recombinant plas- mids that hybridized to the Drosophila probe were isolated napping. The probe for and shown to have identical restriction maps. The restriction BamHI-XhoI fragment map of the insert in pGR24 is shown in Fig. 1.

nuclease assays were
The existence of a physical map of the C. crescentus ya et al. (1) with the genome generated by the ordering of DraI restriction frag-)ns were carried out at ments with respect to the genomic map (10) allowed us to to 56°C for 3 h, with 50 determine the general map position of the dnaK homolog. ,lease S1 digestion was We used the cloned fragment as a probe of Southern blots of 37°C for 30 min. Primer Dral-digested chromosomal DNA separated by pulsed-field [a synthetic oligonucle-gel electrophoresis (10). The cloned DNA hybridized to a ry to nucleotides 433 to single DraI restriction fragment of 305 kb (Fig. 1). Its relative ig. 2). The oligonucleo-position was determined by integrating in both orientations a echnologies, Alameda, plasmid that contained the dnaK gene. This homologous I labeled with y-32P and integration introduced another DraI site, and comparison of total RNA. Annealing the restriction fragments generated by insertion in both piperazine-N,N'-bis(2-orientations allowed the placement of dnaK near the junc-NaCI-5 mM EDTA at tion with the adjacent 105-kb fragment ( Fig. 1 2. DNA sequence of the C. crescentus dnaK gene and a portion of the adjacent dnaJ gene. All of the sequence was determined on both strands and across all restriction sites of the insert in pGR24 shown in Fig. 1. The sequence complementary to the synthetic oligonucleotide (24-mer) is underlined. The inverted repeats in the DNA sequence are indicated by horizontal arrows. enzymes and subcloned into M13mpl8 and M13mpl9, and the DNA sequence was determined by the dideoxy-chain termination method (Fig. 2). DNA sequence analysis revealed an open reading frame coding for a protein that was highly homologous to E. coli DnaK (Fig. 3). A comparison of the predicted amino acid sequence revealed 68% identity with the E. coli DnaK protein (4) and 49% identity with the Drosophila hsp70 protein (18). The dnaK gene could encode a protein of 67.6 kilodaltons. A second open reading frame that did not end in the clone was detected downstream of the dnaK gene (Fig. 2). The TAA stop codon for the dnaK gene was separated from the ATG start codon of the open reading frame by 165 bp. The predicted sequence of the possible 190 amino acids encoded by the open reading frame had 59% identity to the DnaJ protein (5, 27) of E. coli (Fig. 3).
Effect of heat shock on the in vivo transcription of dnaK. The in vivo transcript of the dnaK gene was detected by Northern blot analysis with total RNA prepared from C. VOL. 172, 1990 A D L.aamn]aNSKIIIGIDLGTTNSCVAINDGKTPKVIEN ZE.f col i G * * * * * * * * * * * * * * * * * * * T * * R * L * * *P -A * * * * * * * * Y * * * G V Y Q R G K V E I N A Y CL.clscentus A G A R T T P S V V A F L D G Z RL I G Q P A K R Q A * * * D * * * * * I I * Y T Q * * * T * V * * * * * * * * D~aebiaDQ*N*****Y***T-*S***N*E*.*N*Ṽ VTNPETNPTLFAFAKRLIGRTASDPVVIKDKG R.coli * * * * Q * * * * * * * * * * * * R F Q  crescentus either grown at 30°C or heat shocked at 42°C for the times shown in Fig. 4A. The RNA was electrophoretically separated on formaldehyde agarose gels, transferred onto, nylon filters, and hybridized to a 32P-labeled 3.5-kb BamHI fragment from pGR24. The 3.5-kb fragment hybridized to a 4.0-kb mRNA that was present at low levels in cells growing at normal temperatures (30°C) and was transiently induced approximately 10to 20-fold during heat shock at 42°C, reaching a peak at 15 min. The size of the dnaK gene alone is 1.89 kb. The large size of the transcript and its increased expression upon heat shock, together with the DNA sequence data, suggest that the dnaK and dnaJ genes of C. crescentus could be cotranscribed in a manner similar to that found in E. coli (30,35).
The effect of heat shock on the expression of the dnaK gene was also determined by Si nuclease protection assays.
A probe was constructed by end labeling the BamHI-XhoI fragment shown in Fig. 1. This fragment was hybridized to RNA isolated from cells grown at normal temperatures (30°C) or from cells heat shocked at the times indicated in  (5 ,ug), isolated from cultures grown at 300C (lane 1) and after 5, 10, 15, and 20 min of exposure to 42°C (lanes 2 through 5, respectively), was subjected to Northern blot analysis as described in Materials and Methods. The major transcript at 4 kb is indicated. (B) Nuclease Si protection assays of dnaK transcripts. The RNA (50 ,ug) was from cells growing at 30°C (lane 1) and from cultures shifted to 420C for 10, 15, and 20 min (lanes 2 through 4, respectively). The 5' end-labeled DNA probe was the 1.1-kb BamHI-XhoI fragment shown in Fig. 1. The heat shock-protected RNA fragment of 320 bases and the 280-base protected fragment from cells grown at 30°C are indicated. Fig. 4B. Nuclease Sl-resistant fragments were separated on urea-polyacrylamide gels. RNA isolated from cells grown at normal temperatures yielded a 280-nucleotide protected fragment (Fig. 4B), suggesting that the 5' terminus could lie approximately 150 nucleotides upstream of the dnaK translation start. A faint fragment of 320 nucleotides was also detected in cells grown at 30°C. Upon heat shock, however, this 320-nucleotide protected fragment transiently increased in amount with time of exposure to 42°C and peaked at 15 min. The heat shock transcript mapped to a region 190 nucleotides upstream of the dnaK translation start site.
Identification of the dnaK transcription initiation sites by primer extension. A 24-nucleotide synthetic oligomer complementary to nucleotides 433 to 456 of the dnaK coding sequence shown in Fig. 2 was end labeled with 32P and hybridized to RNA isolated either from heat-shocked cells or from cells grown at normal temperature. The hybrids were extended by reverse transcriptase, and the products were resolved in urea-polyacrylamide gels run next to a sequence (ladder). A single extension fragment was obtained with heat-shocked RNA (Fig. SC, lane 1). The migration of the fragments indicated an initiation site at either the cytidine or guanosine residue at position 236 or 237, respectively (Fig.  5B). Two extension fragments were observed with template RNA from cells grown at normal temperatures (Fig. 5C, lane  2). The less abundant fragment comigrated with the transcript obtained with heat-shocked RNA. The predominant extension fragment corresponded to the smaller transcript detected by Si protection assays (Fig. 4B). The initiation site of this smaller fragment was at the cytidine or guanosine residue at position 279 or 280, respectively (Fig. 5B).
Sequences 5' to the two transcription start sites were compared with E. coli consensus promoter sequences located at positions -10 and -35 (Table 1). Both the sequences at positions -10 and -35 5' to the heat shock transcript (P1) were a close match to the tandem E. coli dnaK P1 and P2 heat shock promoters. However, the sequences 5' to the start of the transcription of the C. crescentus P2 promoter conform, at the -35 region, to the consensus Eu70 promoter, and the -10 region more closely resembles the Eu70 -10 region than it does the Eu32 -10 region.
Temporal control of dnaK expression. The time of appearance of the dnaK transcript was measured in synchronous  Fig. 2) was synthesized and 5' end labeled with [y-52P]ATP. This probe was hybridized for 12 h at 45°C to 50 jig of total RNA from heat-shocked cells (lane 1) or from cells grown at 30°C at 0.85 cell cycle division unit (lane 2). Due to the very strong signal obtained with heat-shocked cells, lane 1 was underexposed. The hybrids were then extended by using reverse transcriptase as described in Materials and Methods.
The sequencing ladder was generated by using the same 24-mer as a primer and M13mpl8 containing the 1.1-kb BamHI-XhoI fragment (coding strand). The sequence shown is the complement. The potential transcription start sites are indicated by arrows. cultures grown at 300C with the Si nuclease protection assay (Fig. 6A). Total RNA isolated at different times during the cell cycle was hybridized to a 32P-labeled 1.1-kb BamHI-XhoI probe (Fig. 1) and then treated with Si nuclease. The 280-nucleotide protected fragment was present at high levels between 0.25 and 0.35 division units, coincident with the initiation of DNA replication. After a period of low transcript levels, the RNA fragment was again detected in large amounts near the end of the cell cycle, just before the initiation of DNA replication in the progeny stalked cell. The 320-nucleotide fragment, which was protected by RNA isolated from heat-shocked cells, was also detected in these 3056 C .  experiments, but at lower levels. At normal physiological temperatures the transcript from the heat shock promoter P1 was temporally controlled, analogous to the transcript from the P2 promoter.
To determine whether the sequences 5' to the start of the dnaK gene were sufficient to control its temporal expression, the 1.35-kb BamHI-PstI fragment containing the entire upstream region plus 370 bp of the coding region of the dnaK gene ( Fig. 1) was subcloned into the transcription reporter vector pUCD615. This vector contains a promoterless lux operon from Vibrio fisheri (16). The expression of the lux genes fused to the dnaK promoter was monitored by measuring photon emission as described in Materials and Methods. Expression of the transcription fusion directed by the dnaK promoter was cell cycle regulated (Fig. 6B). The promoter region of dnaK appeared to initiate transcription of the chimeric gene during the swarmer to stalk transition and again in the predivisional cell just before cell division. In each instance the gene was expressed immediately before the initiation of DNA replication. DISCUSSION hsp70 genes are present in a wide variety of species. Genes related to Drosophila hsp70 have been isolated from organisms as diverse as E. coli, Saccharomyces cerevisiae, maize, chickens, and humans (21). A hig-h degree of similarity exists among these genes and their encoded proteins. A comparison of the protein-coding region of the E. coli and Drosophila genes, for instance, reveals 57% identity at the nucleotide level (4). Using a Drosophila hsp70 gene probe, we isolated a fragment of C. crescentus DNA that appeared to encode the DnaK heat shock protein: Sequence analysis reyealed that the predicted amino acid sequence of the C. crescentus DnaK protein was 68% identical to E. coli DnaK and 49% identical to Drosophila hsp70. Sequence analysis of the region downstream of the dnaK gene revealed a coding sequence for a peptide that was 59o identical to E. coli DnaJ.
The heat shock response in E. coli is regulated by the rpoH gene product, a32, which directs RNA polymerase to initiate transcription from heat shock promoters at all temperatures (7). The increased synthesis of heat shock proteins after a shift to higher temperatures is caused by increased transcription initiation from heat shock gene promoters by 3 RNA polymerase. Recent experiments have demonstrated that the concentration of E&32 transiently increases after a shift to a high temperature, due to an increase in rpoH mRNA level occurring mainly through a posttranscriptional mechanism (11,14). Three heat-inducible promoters for the operon containing dnaK and dnaJ genes were identified both in vivo and in vitro in E. coli. These promoters are also used at normal temperatures. In C. crescentus, the dnaK gene also had two transcription start sites, but only one was heat inducible.
The promoter corresponding to the transcript that increased in abundance during the heat shock response, P1, had a -10 region that conforms to the consensus -10 region for heat shock promoters of E. coli (4), and its -35 region fits reasonably well with the consensus -35 region of E. coli heat shock promoters (Table 1). These results suggest that C. crescentus contains a minor sigma factor similar to a32 of E. coli. A heat shock protein of 37 kilodaltons was previously detected in C. crescentus by immunoprecipitation with anti-RNA polymerase antibody (29). This protein is a candidate for the C. crescentus heat shock sigma factor but awaits biochemical analyses with the purified protein.
The P2 promoter, which appears to conform to the EU70 promoter and the U70 promoters found in front of some C. crescentus genes, was active only during growth at physiologic temperature (30°C) but was under temporal control. All of the regulatory sequences required for temporal control of dnaK transcription appeared to be within the 820 bp 5' to the start of transcription. This conclusion is supported by the observation that this 5' regulatory region and 370 bp of dnaK coding sequence fused to a promoterless lux operon resulted in the same temporal expression of the lux activity as the expression of the chromosomal dnaK gene. The C. crescentus dnaK gene was transcribed just preceding the S phase during the transition from swarmer to stalked cells and again in late predivisional cells just before the initiation of DNA replication in the progeny stalked cell. These results confirm previous observations that the C. crescentus dnaK protein is synthesized at defined times in the cell cycle (13).
Transcription at the putative heat shock promoter under normal physiological conditions also varied as a function of the cell cycle, showing an increase at the transition from swarmer to stalked cells and again when cells were beginning to divide (0.85 to 1.0 division units). There are at least two possible explanations for this. In other systems the increase in transcription from heat shock promoters is mediated through increases in the level of cr32. It may be that the comparable heat shock sigma factor gene is under cell cycle modulation and that other heat shock proteins are also increased at this point during the cell cycle. More likely, cis-acting sequences that contribute to temporally controlled VOL. 172, 1990 3057 modulation of gene expression affect the rate of dnaK transcription by both the constitutive but low levels of the heat shock sigma factor and the sigma 70 factor.
The function of DnaK in C. crescentus is not known, although there are a number of possible roles for this heat shock protein during the normal cell cycle. DnaK and other heat shock proteins might participate in global control of development in C. crescentus. This function may be mediated through stability and proteolysis of cell-type-specific proteins. Alternatively, the DnaK protein might be required for some aspect of C. crescentus DNA replication. It is known that the E. coli DnaK and DnaJ proteins are required for successful initiation of phage lambda DNA replication (19), and recently a mutant allele of dnaK has been shown to cause conditionally defective initiation of E. coli DNA replication (31). Maximal transcription of dnaK in C. crescentus reflects the time of initiation of new rounds of DNA replication; in fact, the DnaK protein specifically segregates to the progeny stalked cell, in which a new round of DNA replication occurs (29). These results are analogous to that seen with the cell-cycle-controlled transcription of the human hsp7O gene in HeLa cells (23). Thus, the expression of a highly conserved heat shock protein in such diverse cell types as a bacterial cell and a human cell shows parallel cell cycle control under normal physiological conditions.