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Journal of Bacteriology, May 2007, p. 3751-3758, Vol. 189, No. 10
0021-9193/07/$08.00+0     doi:10.1128/JB.01722-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Expression Analysis of Multiple dnaK Genes in the Cyanobacterium Synechococcus elongatus PCC 7942{triangledown} ,{dagger}

Masumi Sato, Kaori Nimura-Matsune, Satoru Watanabe, Taku Chibazakura, and Hirofumi Yoshikawa*

Department of Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

Received 8 November 2006/ Accepted 27 February 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We analyzed the stress responses of three dnaK homologues (dnaK1, dnaK2, and dnaK3) in the cyanobacterium Synechococcus elongatus PCC 7942. A reporter assay showed that under stress conditions the expression of only the dnaK2 gene was induced, suggesting a functional assignment of these homologues. RNA blot hybridization indicated a typical stress response of dnaK2 to heat and high-light stress. Primer extension mapping showed that dnaK2 was transcribed from similar sites under various stress conditions. Although no known sequence motif was detected in the upstream region, a 20-bp sequence element was highly conserved in dnaK2; it was essential not only for the stress induction but also for the basal expression of dnaK2. The ubiquitous upstream localization of this element in each heat shock gene suggests its important role in the cyanobacterial stress response.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Living organisms are equipped with mechanisms that allow them to survive under various environmental stresses by the rapid induction of specific sets of genes such as heat shock genes encoding heat shock proteins (Hsps). DnaK (Hsp70), one of the major Hsps, is a member of molecular chaperones essential for maintaining and restoring protein homeostasis. Although Hsp70 has been identified in various organisms, the presence of a multigene family of Hsp70 is rare in prokaryotes. With the exception of a few groups, almost all prokaryotes whose genomes have been sequenced contain a single dnaK gene.

Cyanobacteria are photosynthetic prokaryotes that have developed an oxygen-producing photosynthetic system similar to that of higher plants. They constitute a diverse group of organisms, and the entire genomes of many strains have been sequenced. Interestingly, their genomes contain multiple dnaK genes. We previously identified three dnaK homologues (dnaK1, dnaK2, and dnaK3) in the genome of cyanobacterium Synechococcus elongatus PCC 7942 (28, 29) and reported that DnaK2 and DnaK3 are essential for normal growth and that substantial amounts of DnaK3 are localized on the surface of the thylakoid membrane. On the other hand, DnaK1 and DnaK2 were primarily found in the cytosol (27), suggesting the functional diversity of these DnaK proteins. In most eukaryotes, Hsp70 is identified as a multigene family; its existence in various cellular compartments (11, 20) indicates that each chaperone functions within multiple cellular compartments. Because it remained unclear how the functions of these Hsp70 proteins in the same cellular compartment are assigned, we studied the specific function of cyanobacterial DnaK. The level of each DnaK protein varies in response to heat shock: the synthesis of DnaK2 increased upon temperature upshift, whereas the synthesis of DnaK1 and DnaK3 was not affected; rather, the level of DnaK1 protein decreased (26). Based on these observations, we postulated that the expression of these dnaK genes is regulated differently.

In the present study we first examined the functional assignment of these DnaK under various growth conditions and then attempted to profile their expression under various stress conditions. Second, we examined the gene regulation mechanism(s) of dnaK2 because, among the three dnaK genes, it was the only one to exhibit a typical stress response. The regulation of heat shock gene induction has been studied in prokaryotes. In Escherichia coli and Bacillus subtilis, a sigma factor-related stress response governs many genes encoding stress protective factors (8, 13, 41). On the other hand, negative regulatory mechanisms are more diverse. Several repressor proteins and their binding sites have been reported. For example, the HrcA repressor and its binding site CIRCE (for controlling inverted repeat for chaperone expression) have been identified in many gram-positive bacteria (25), and the HspR repressor and cis element HAIR (for HspR-associated inverted repeat) regulate the dnaK operon and clpB in Streptomycetes and Mycobacteria (5, 35). Moreover, the CtsR system in B. subtilis (9, 10) and the RheA repressor in S. albus (32) have been reported. However, despite its typical stress response, in Synechococcus dnaK2 we could find no known regulatory sequences upstream; this suggested a novel stress induction mechanism. Since dnaK genes are a highly conserved ubiquitous group of heat shock genes, we were interested in discerning their regulation mechanism(s). Here we report a unique regulatory region of dnaK2, discuss the possibility of trans factor involvement, and suggest that it may represent a unique regulation mechanism of heat shock genes among prokaryotes.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains, plasmids, and culture conditions. Unless otherwise indicated, Synechococcus sp. strain PCC 7942 wild-type and its derivatives were grown photoautotrophically at 30°C in BG-11 medium (7) under bubbling with air and continuous illumination (40 µE m–2 s–1). Where appropriate, the medium was supplemented with kanamycin or spectinomycin at a final concentration of 10 or 40 µg/ml, respectively. The plasmids and strains are listed in Table 1. The construction of the pDrZ–, pNSZ–, and pNSbgaB+ plasmids is described below.


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  		TABLE 1. Bacterial strains and plasmids used in this study

 
Construction of the pDrZ- plasmid. The plasmid pDrZ– was constructed by ligating the 3.8-kb lacZ-kanamycin-resistant (Kmr) gene fragment with the 3.4-kb BamHI and ScaI restriction fragment of pTrc99A/X (Pharmacia) containing the origin of replication for E. coli and the lacI and trc promoters. For the generation of the lacZ-Kmr gene fragment, we used the recombinant PCR method (15). Secondary-stage PCR products were generated by using the primary PCR fragments containing the 3-kb lacZ fragment (using the primers BamlacZ-f and KMlacZ-r, see Table S1 in the supplemental material) and the 800-bp Kmr gene fragment (primers lacZKM-f and ScaKM-r) whose ends overlap. The templates for the lacZ and Kmr gene fragments were pMutin2 (39) and pUC4K (Pharmacia), respectively. pDrZ- has two multicloning sites, CS1 (derived from pTrc99A/X) and CS2 containing SalI and SphI sites derived from the ScaKM-r primer. This plasmid replicates only in E. coli.

Construction of the strain carrying a dnaK-lacZ fusion in the dnaK locus. Fragments containing lacIq and Ptrc-dnaK derived from pTrcDk1, pTrcDK2, and pTrcDK3 plasmids (26) were cloned into the pNS1 plasmid (27). The resulting plasmids were used to transform wild-type Synechococcus to obtain spectinomycin-resistant strains carrying the IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducible dnaK gene at the neutral site (6) of the chromosome (NBC101, NBC102, and NBC103; Table 1). Next, PCR fragments generated from the wild-type chromosome and containing 600 bp of upstream (using primers rr-f2 and rr-r2; see Table S1 in the supplemental material) and downstream (primers Salds-f and Sphds-r) sequences of each dnaK gene were cloned into CS1 and CS2 of plasmid pDrZ– (see Fig. S1 in the supplemental material). The resulting plasmids were used to transform strains NBC101, NBC102, and NBC103 individually to obtain the kanamycin- and spectinomycin-resistant strains carrying each dnaK-lacZ fusion at the original dnaK locus (NBC104, NBC105, and NBC106; Table 1). NBC104 and NBC106 could grow in medium without IPTG by leaky expression from the trc promoter, whereas NBC105 required 0.1 mM IPTG in the medium.

ß-Galactosidase assays. Synechococcus cells harboring the dnaK-lacZ fusion were grown at the indicated conditions and culture aliquots were withdrawn for ß-galactosidase assay (3); activities are expressed as Miller units. When using the bgaB fusion (see below), the assays were carried out at 60°C.

Northern blot analysis. Total RNA was isolated from Synechococcus cells by using hot phenol as previously described (31). For all RNA blot hybridizations, equal amounts of RNA were resolved by electrophoresis on 1.0% (wt/vol) agarose gels containing 5.5% (wt/vol) formaldehyde and subsequently blotted onto Hybond-N+ membranes (Amersham Bioscience). The digoxigenin-labeled, dnaK2-specific RNA probe was prepared with a DIG RNA labeling kit SP6/SP7 (Roche Diagnostics) using the primer pair described in Table S1 in the supplemental material. Hybridization was according to the manufacturer's instructions. Signals were detected on the Lumi-Imager F1 (Boehringer Mannheim, Mannheim, Germany) and visualized with the LumiAnalysit version 3.0 program; brightness and contrast were adjusted to enhance the images.

Primer extension mapping. Total RNA extracted from cells grown under high-light or heat (100 µg) or under high-NaCl or normal growth conditions (1,000 µg) was mixed with 1 pmol of the IRD800-labeled primer (ALOKA, Tokyo, Japan), which is complementary to the 100-bp downstream portion of the initiation codon of dnaK2 (Table S1 in the supplemental material). The RNA-primer mix was heated to 70°C for 5 min and allowed to cool at room temperature. Reverse transcription was with the BcaBEST RNA PCR kit version 1.1 (TaKaRa, Shiga, Japan). The samples were precipitated with ethanol and resuspended in 100 µl of H2O, 100 µl of 0.6 N NaOH was added, and this was followed by 30 min of incubation at 65°C to degrade the RNA. After neutralization by adding 100 µl of 0.6 N HCl, the samples were precipitated with ethanol. The reverse-transcribed samples and the products of dideoxyribonucleotide sequencing reactions, performed with the same primer and a ThermoSequenase cycle sequencing kit (USB), were dissolved in 4 µl of stop solution provided by the supplier. After heat denaturation, electrophoresis was carried out on 4% urea-polyacrylamide sequencing gels by using a DNA sequencer (model 4000L; LI-COR).

Construction of the strain carrying a dnaK2-lacZ or dnaK2-bgaB fusion at the neutral site. The Synechococcus strain carrying the dnaK2-lacZ translational fusion or the dnaK2-bgaB transcriptional fusion at the neutral site was constructed by using plasmid pNSZ– and pNSbgaB+, respectively. These plasmids were based on pAM990 (19). The lacZ gene fragment lacking its Shine-Dalgarno (SD) sequence was PCR generated using the primers BgllacZ-fnoSD and KpnlacZ-r (Table S1 in the supplemental material) and pAM990 as a template. The bgaB gene fragment was amplified with the primer pair BgaB-f and BgaB-r (Table S1 in the supplemental material) to include the SD sequence at its 5' end; the pDLd plasmid (24) was the template. The lacZ gene and its flanking region were deleted from pAM990 by digestion with BglII and KpnI; it was then ligated with those PCR fragments to obtain pNSZ– and pNSbgaB+. These plasmids replicate only in E. coli. A PCR fragment amplified with the primers K2up1K and K2rr-r2 (see Table S1 in the supplemental material) was cloned into pNSZ– and pNSbgaB+, and these plasmids were used to transform Synechococcus wild-type (NBC100; Table 1) to construct strains NBC107 and NBC112 (Table 1), respectively. Similarly, NBC108 and NBC109 were constructed using the primers K2conf1 and K2rr-r2 and the primers K2conf2 and K2rr-r2, respectively. Two PCR fragments generated with primers K2up1K and K2-con1 and primers K2-con2 and K2rr-r2 (see Table S1 in the supplemental material) were recombined, and the products were then cloned into pNSZ– and pNSbgaB+ to obtain strains NBC111 and NBC113, respectively. PCR fragments for NBC110 were generated by using primers K2up1K and K2con+pro1 and the primers K2con+pro2 and K2rr-r2. For construction of NBC140 containing point-mutated MARS (for multistress associated regulatory sequence), PCR fragment generated using primers K2modi-con1 and K2rr-r2 (see Table S1 in the supplemental material) was cloned into pNSZ–, and the resultant plasmids were used to transform wild-type Synechococcus. Similarly, NBC141, NBC142, NBC143, NBC144, and NBC145 were constructed by using the primers K2modi-con-2, K2modicon-3, K2modicon-4, K2modicon-5, and K2modicon-6 (see Table S1 in the supplemental material) as the forward primer and the primer K2rr-r2 as the respective reverse primer. For PCR of the Synechococcus dnaK2 region, the wild-type chromosome was the template.

Database. Synechococcus and other cyanobacterial gene sequences were retrieved from the CYORF Cyanobacteria Gene Annotation Database (http://cyano.genome.jp/).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression profiles of the three dnaK genes in Synechococcus under stress conditions. To analyze the expression of the three Synechococcus dnaK genes under stress conditions, we used the lacZ gene as the reporter gene. Figure S1 in the supplemental material shows the insertion of a translational lacZ gene fusion at each original dnaK locus and the construction of the ectopically expressed dnaK gene at the neutral site (6) of the Synechococcus chromosome. Translational ß-galactosidase fusions with the N-terminal region (40 amino acids) of DnaK and a selectable marker were flanked by upstream and downstream sequences of each dnaK on a nonreplicative plasmid (pDrZ–). Since dnaK2 and dnaK3 are essential for normal growth (26), transformation was carried out with strains that have a corresponding dnaK gene at the neutral site of the chromosome (strains NBC101, NBC102, and NBC103; see Materials and Methods) rather than with wild-type Synechococcus. In case these dnaK genes are autoregulatory, it is possible that the expression levels of Ptrc-dnaK in the neutral site have a feedback effect on lacZ activity. We investigated the effect of dnaK overexpression by the addition of IPTG, which induces the expression of Ptrc-dnaK (see Materials and Methods). The ß-galactosidase activities of strains NBC104, NBC105, and NBC106 grown in medium supplemented with IPTG at a final concentration of 2 mM were identical to those without or with 0.1 mM (NBC105) IPTG (data not shown). These results suggest that Synechococcus dnaK genes are not autoregulatory and that the expression of reporters cannot be influenced by Ptrc-dnaK.

The ß-galactosidase activity was measured in cells exposed to high-light (Fig. 1A) or high-salt stress (Fig. 1B). dnaK2-lacZ expression was clearly induced under both stress conditions; on the other hand, dnaK1 and dnaK3 showed little response. dnaK2 responded transiently to high-light conditions, reaching a maximum of expression after 120 min of exposure (Fig. 1A); a quick and weak response was observed under the high-NaCl condition, and induction seemed to continue for up to 120 min (Fig. 1B). To discern whether N-terminal fusion of DnaK affects the stress response, we also constructed a strain that carries lacZ fused to the initiation codon of each dnaK (no 40 amino acids). The results were the same as in Fig. 1 (data not shown), demonstrating that only dnaK2 exhibited remarkable induction under both stress conditions.


Figure 1
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  		FIG. 1. Expression analysis of the three dnaK genes under high-light and salt stress conditions. The cells were grown under normal growth conditions, exposed to high light (400 µE m–2 s–1) for 300 min (A), or treated with high NaCl (0.5 M) for 120 min (B), and the ß-galactosidase activity was assayed. Time zero indicates the point at which cells (optical density at 750 nm of ~0.6) were exposed to each stress type. Assays with strains NBC104, NBC105, and NBC106 (cells that express dnaK1, dnaK2, and dnaK3 promoter-lacZ) are represented by closed circles, triangles, and squares, respectively. For the dnaK2-lacZ assay in panel B, the same volume of H2O was added as a control (indicated by open triangles with dashed lines). The indicated activity values are the means of measurements from at least three independent experiments, and the standard deviations are shown for the assay of dnaK2 (NBC105).

 
RNA blot hybridization of the dnaK2 gene under various stress conditions. We measured the amount of dnaK2 mRNA under conditions of heat, high light, high salt, and osmotic stress (Fig. 2). dnaK2 responded to all types of stress; the response to high-light stress was intense and moderate to heat, high-salt, and high-osmolarity stress. Transcription enhancement was maximal within 10 to 15 min of exposure to heat or high light. The accumulated level of transcripts was much lower under heat than high-light stress (Fig. 2A and B), and the response to high-salt and hyperosmotic stress was delayed (Fig. 2C and D).


Figure 2
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  		FIG. 2. RNA blot analysis of dnaK2 under various stress conditions. Total RNA was isolated from cells subjected to high-light (400 µE m–2 s–1) (A), heat (45°C) (B), NaCl (0.5 M) (C), and hyperosmotic (0.5 M sorbitol) (D) stress. For RNA blot hybridization, equal amounts of RNA were hybridized with a dnaK2-specific probe (top panels). Quantification of the dnaK2 transcripts is shown below each RNA blot. The indicated transcript amounts are the means of measurements from at least three independent experiments; they are expressed as values relative to the levels obtained under normal conditions (before exposure to each type of stress). Ethidium bromide-stained rRNA was the loading control (bottom panels). Time zero is as in Fig. 1.

 
Analysis of the upstream region of dnaK2. Because it was the only stress-responsive dnaK in Synechococcus, we studied the upstream region of the dnaK2 gene. Figure 3 shows the results of primer extension mapping of the dnaK2 transcript during the early stage of various stress responses. Although it identified several transcription start sites of dnaK2, upstream of these sites there was no sequence homology with any consensus promoter sequences known in E. coli or B. subtilis (Fig. 3). There was no difference in the transcription start sites under the different stress conditions. It is possible that the downstream sites (–59 A and –60 A) observed under heat and high-light stress were derived from the cleavage of a hairpin structure (Fig. 3). To determine the transcription start sites with confidence, we carried out primer extension analysis with another primer, which is complementary to the 50-bp downstream of the initiation codon of dnaK2. We obtained identical results with above-mentioned experiments (data not shown).


Figure 3
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  		FIG. 3. (A) Identification of the 5' end of the dnaK2 transcript. Total RNA was isolated from cells grown under normal growth conditions (lane 1), and cells were exposed for 15 min to high-NaCl (0.5 M) (lane 2), high-temperature (45°C) (lane 3), and high light (400 µE m–2 s–1) (lane 4). Lanes T, G, C, and A show the dideoxy-sequencing ladder using the same primer as for the reverse transcription. The numbers on both sides of a sequence indicate the positions relative to the translation initiation site. The sequence represents the upstream region of dnaK2 with the coding region shown in italics. The box contains the ATG start codon. Putative transcription start sites of dnaK2 are indicated in boldface with asterisks; arrows represent inverted repeat sequences. Numbers and arrowheads on the sequence indicate positions relative to the putative transcriptional start sites. The highly conserved element is shaded (see Fig. 4).

 
Conserved sequence identified in upstream region of cyanobacterial hsp genes. We were unable to detect homology with sequences known to be involved in the stress response of prokaryotes. Interestingly, we identified a 20-bp sequence element that was highly conserved among cyanobacterial dnaK2 (Fig. 3 and 4). The consensus sequence 5'-A/CG/TGTTCGGGAANCNCNCCTT/C-3' was identified from the alignment of the upstream sequences of 11 dnaK2 orthologs. Moreover, sequences similar to this element were also observed in the within 200-bp upstream regions of other stress-related genes in cyanobacteria (Fig. 4). The element was therefore designated MARS, for "multistress-associated regulatory sequence." The consensus sequence of MARS is illustrated in Fig. 4, although the conservation of the sequence of hspA or clpC orthologs is lower than that of dnaK2, groES, or groEL2.


Figure 4
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  		FIG. 4. Alignment of the conserved element identified in upstream regions of stress-related genes in cyanobacteria. MARS are retrieved from the upstream of indicated orthologs and aligned. The open reading frame identification of each gene is listed in Table S2 in the supplemental material. Numbers on the sides of each sequence indicate the positions relative to the translation initiation site of each gene. In each ortholog, the nucleotides conserved in more than half of the strains are shaded; completely conserved nucleotides are shown in black. syf, Synechococcus elongatus PCC 7942; syw, Synechococcus sp. strain WH 8102; pma, Prochlorococcus marinus SS120; pmm, Prochlorococcus marinus MED4; pmt, Prochlorococcus marinus MIT 9313; syn, Synechocystis sp. strain PCC 6803; ana, Anabaena sp. strain PCC 7120; tel, Thermosynechococcus elongatus; gvi, Gloeobacter violaceus PCC 7421; cya, Cyanobacteria Yellowstone A-Prime; cyb, Cyanobacteria Yellowstone B-Prime.

 
Effects of different alterations in the upstream sequence of dnaK2 on the expression of reporter genes under high-light and heat stress. To identify the regulatory region of dnaK2, we constructed a series of Synechococcus reporter strains in which the expression of reporter genes was driven by upstream fragments of dnaK2. We used the bgaB gene encoding thermostable ß-galactosidase as the reporter gene in heat-stress experiments. Because the activity of the bgaB translation product was relatively low (Fig. 5C), we used the lacZ gene in high-light experiments (Fig. 5B). Each fragment shown in Fig. 5A was translationally fused to lacZ in the same construction as in Fig. 1; transcriptional gene fusions were used in heat stress experiments because the additional N-terminal region had a strong negative effect on bgaB activity. We first cloned the region from –928 to +192 relative to the putative transcription start site; this induced reporter expression under both high-light and heat stress (strain NBC107 and NBC112 in Fig. 5). The difference in the expression patterns of the lacZ and bgaB fusions may be attributable to differences in the half-life of these enzymes. The strain with the region from –49 to +192 that contained only the MARS element and its downstream region (NBC108 in Fig. 5B) exhibited a high-light response similar to that of strain NBC107, but basal expression was reduced to about one-half (Fig. 5B). To study the role of MARS, we constructed a strain that carried the region from –928 to +192 but lacked MARS (NBC111 in Fig. 5B). Since the MARS sequence includes a putative promoter region at –35 relative to the transcription start sites, we constructed partial MARS mutants (NBC109 and NBC110 in Fig. 5A) to rule out the possibility that a full-length MARS deletion resulted in altered promoter structure. As shown in Fig. 5B, in strains NBC109, NBC110, and NBC111, deletion of the MARS element, including an 8-bp deletion at its 5' end, produced a defect in basal expression and hampered the high-light stress response, suggesting that full-length MARS is essential for the basal expression of dnaK2. Under heat stress, the bgaB assay yielded similar results (NBC113 in Fig. 5C). For more detailed studies on the involvement of MARS in dnaK2 expression, we examined the effect of point mutation of MARS. As shown in Fig. 6A, there are inverted repeat sequences within MARS derived from a variety of dnaK2 genes. We therefore altered the highly conserved nucleotides in inverted repeat region (Fig. 6B, indicated in boldface in NBC108). The strain NBC141 whose –46T in MARS is changed to G (Fig. 6B) exhibited high-light response and about a half of the ß-galactosidase activities of NBC108, whereas the expression of other mutants were markedly reduced (Fig. 6C). The substituted nucleotide of NBC141 (–46T) differs from those of the other nucleotides analyzed (–47G, –44C, –43G, and –42G) in that it was conserved incompletely among 11 dnaK2 orthologs (Fig. 6A and B).


Figure 5
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  		FIG. 5. Effect of various upstream regions of dnaK2 on dnaK2-lacZ and dnaK2-bgaB expression under high light and heat stress in Synechococcus. (A) Schematic drawing of various upstream fragments of dnaK2 tested. Each upstream fragment indicated in panel A was fused individually to the lacZ gene in pNSZ– (Table 1) or the bgaB gene in pNSbgaB+ (Table 1). The white, striped, and shaded bars indicate the region of aroE (the open reading frame located upstream of dnaK2), the highly conserved element, and the N-terminal coding sequence of dnaK2, respectively. Numbers indicate positions relative to the transcriptional start site (the precise upstream sequence is indicated in Fig. 3). (B and C) Homologous recombination of these plasmids at the neutral site of the chromosome resulted in the reporter constructs illustrated in panels B and C, respectively. In panel C, SD indicates the SD sequence of the bgaB gene derived from pNSbgaB+. Strains NBC107 and NBC112 contain the lacZ and bgaB genes, which are fused to the region from –928 to +192 relative to the putative transcription start site of dnaK2, respectively. In strain NBC108, the region from –49 to +192 (MARS and its downstream region) is fused to lacZ gene. Similarly, strains NBC109 and NBC110 have the region from –41 to +192 (partial MARS mutant and its downstream region) and –928 to +192 containing partial MARS mutant, respectively. Strains NBC111 and NBC113, whose reporter genes are lacZ and bgaB, respectively, carry the region –928 to +192 lacking MARS. The ß-galactosidase activity of cells grown under normal growth conditions (time zero) or high-light (400 µE m–2 s–1 [B]) or heat (45°C [C]) stress conditions was measured as in Fig. 1.

 

Figure 6
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  		FIG. 6. Effect of base substitution of MARS on dnaK2-lacZ expression. (A) An alignment of MARS for dnaK2 is redrawn from Fig. 4. Arrows represent inverted repeat sequences. The nucleotides conserved completely among 11 sequences are shaded with asterisks. (B) Base substitutions of Synechococcus MARS. Substituted nucleotide are boxed and indicated in boldface. The numbers on the sequences indicate positions relative to the transcriptional start site. The structures of strains NBC140 to NBC145 are the same as that of NBC108 (Fig. 5 and 6) except for the mutated points. (C) ß-Galactosidase activity measured as in Fig. 5.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Reporter assay using the lacZ gene revealed dnaK2 to be the only hsp70 gene that showed upregulated expression under high-light and NaCl stress conditions (Fig. 1). This, together with our finding that only the synthesis of DnaK2 increased upon temperature upshift (26), suggests that dnaK2 is unique among the three dnaK genes exhibiting a stress response, although they are expressed in the same cellular compartment. This observation strongly supports the functional assignment of these DnaK proteins. Its amino acid identity (DnaK1, 50.5%; DnaK2, 57.1%; DnaK3, 51.5%) and complementation analysis (26) indicated that among the three DnaK proteins, DnaK2 is most similar to the protein of E. coli. Moreover, DnaK2 subgroup members appear to be strictly conserved in all cyanobacteria lineages, and phylogenetic analysis of DnaK proteins (4) showed that they represent the only DnaK chaperone retained in algal and plant plastids. This suggests that DnaK2 has the most essential Hsp70 functions. We postulate that DnaK2 has the functions of a general molecular chaperone, e.g., protein folding, oligomer assembly, and stabilization of the protein structure. However, as stress might affect photosynthesis, we cannot exclude the possibility that DnaK2 has specific functions in cyanobacteria. Although Synechococcus DnaK1 is similar to the DnaK1 of the halophytic cyanobacterium Aphanothece halophytica (4, 14, 18), we did not observe marked induction under high-NaCl conditions (Fig. 1B). Since both osmotic pressure and downshifted temperature have similar effects on membrane lipids, we profiled the expression of each dnaK gene under low-temperature (16°C) conditions. Like dnaK2 and dnaK3, dnaK1 was not induced by cold stress (data not shown). DnaK3 is thought to be involved in the translational process on the thylakoid membrane (17), although its constitutive expression was observed under growth conditions and high-light stress (Fig. 1A), suggesting that in cyanobacteria the function of DnaK3 is independent of light intensity. Studies are under way in our laboratory to identify the significance of multiple dnaK genes in cyanobacteria.

We confirmed our reporter gene assay results by Northern blot analysis. dnaK2 responded to heat and high osmotic stress and to high-light and salt stress (Fig. 2). Its response to heat stress was much weaker than to high-light stress. The difference in the response levels suggests differences in the severity of the adverse effects of these stresses or the necessity for a molecular chaperone. Under high-NaCl and hyperosmotic stress conditions, the response was much slower and occurred in phases. In Synechocystis sp. strain PCC 6803, the salt stress response has been well studied; a transient efflux and subsequent uptake of potassium ions occurred within 2 h of salt treatment. Thereafter, the potassium concentration decreased, and the content of compatible solutes continued to increase for prolonged periods (21). We postulate that these distinct phases of the long-term salt stress response may result in the delayed and complex expression of dnaK2. Under high-NaCl and high-sorbitol conditions, the cytoplasmic volume of Synechococcus and Synechocystis strains decreased (1, 2, 16). However, the degree of volume reduction was markedly different with the two types of stress, possibly because the plasma membrane is sorbitol impermeable (2) and ion permeable, suggesting that NaCl and sorbitol stress may exert different effects on the cellular physiology. Our observation of a lower degree of dnaK2 induction by sorbitol than NaCl may reflect this difference.

The results of Northern blotting suggest a multistress response for Synechococcus dnaK2. Since dnaK2 exhibited the typical stress response to heat and high-light stress, we examined the upstream region, which might be involved in the regulatory mechanism(s) underlying dnaK2 induction under these stress conditions. Primer extension mapping showed that dnaK2 was transcribed from similar sites under different stress conditions, although no known promoter or regulatory sequence motif, e.g., CIRCE (25) or HAIR (12), was detected in the upstream region (Fig. 3). This was an unexpected finding since the regulatory mechanism of dnaK has been studied intensively. Although the CIRCE element was found in the 5'-untranslated region of the Synechococcus groESL operon (40), the heat induction of groESL persisted in the Synechocystis hrcA mutant (23), suggesting a unique regulatory system of hsp genes in cyanobacteria. We identified a 20-bp sequence element that was highly conserved among cyanobacterial stress-related genes, including dnaK2 (Fig. 3 and 4). In several strains, we observed inverted repeat sequences in this element (Fig. 6A), possibly reflecting a recognition site of the trans factor. This conserved element, designated MARS, was essential not only for the stress induction but also the basal expression of Synechococcus dnaK2 (Fig. 5). Among the nucleotides that compose MARS, substitution of –46T conserved incompletely still exhibited a high-light stress response and basal expression, whereas changes in the nucleotides (i.e., –47G, –44C, –43G, and –42G) completely conserved among 11 dnaK2 orthologs resulted in a significant decrease in basal expression (Fig. 6). Since the –43G and –42G positions of dnaK2 MARS are not composed of inverted repeats, highly conserved nucleotides may be responsible for dnaK2 expression rather than the inverted repeat structure. It remains uncertain whether MARS contributes to recognition via a basic transcriptional machinery or via other regulatory systems. The ubiquitous upstream localization of this element in each stress-related gene raises the possibility that MARS contributes to a global stress response in cyanobacteria. Our ongoing study of the role(s) of MARS and of interacting factors may reveal a novel regulatory mechanism for hsp genes.

To identify trans factors involved in the heat induction of dnaK2, we constructed deletion mutants of groESL and/or groEL2, the molecular chaperone, because hsp70 and hsp60 genes are known to influence each other's expression (22, 36, 38). We also constructed {Delta}hik34 (Synpcc7942_1517) and {Delta}rre1 (Synpcc7942_1860) mutants, factors of the two-component type system. Hik34, known to regulate the expression of the dnaK2 via its cognate response regulator Rre1 under salt and osmotic stress (30, 33), is reportedly a sensor or signal transducer in sensing the heat stress of certain hsp genes (34, 37) in Synechocystis. Although we could not segregate completely the disruptant of groEL and rre1, no decrease in the induction of dnaK2 was observed in these Synechococcus disruptants. We posit that other two-component factor(s) or other regulatory systems are involved in its heat induction. We performed similar experiments with sigma factor deletion mutants. Northern blot analysis of dnaK2 using nine disruptants, except the major sigma factor rpoD1, confirmed distinct heat induction in all deletion mutants (data not shown). It remains to be determined whether RpoD1 governs the transcription of dnaK2, or whether several sigma factors have contributory roles.

In summary, our findings based on primer extension, analysis of the upstream region, and mutant analysis have led us to conclude that stress induction of the Synechococcus dnaK2 gene is regulated by a previously unknown mechanism. Additional factors such as the trans factor may provide clues to the regulatory system, including the role of MARS, in the dnK2 gene, the only stress-responsive dnaK gene.


   ACKNOWLEDGMENTS
 
We are indebted to Mamoru Sugita at Nagoya University for providing the genome sequence of Synechococcus sp. strain PCC 6301 prior to its publication. We are grateful to Kan Tanaka at the University of Tokyo for providing the Synechococcus {Delta}rpoD2, {Delta}rpoD3, and {Delta}rpoD4 mutant strains and to Shingo Ozawa for technical assistance.

This study was supported by a Grant-in-Aid for Scientific Research on Priority Area C (Genome Biology) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan. Phone: 81 (3) 5477 2758. Fax: 81 (3) 5477 2668. E-mail: hiyoshik{at}nodai.ac.jp Back

{triangledown} Published ahead of print on 9 March 2007. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Journal of Bacteriology, May 2007, p. 3751-3758, Vol. 189, No. 10
0021-9193/07/$08.00+0     doi:10.1128/JB.01722-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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