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Elevated temperatures as well as many other stresses such as high concentrations of ethanol, arsenite, and heavy metals elicit a physiological response resulting in the synthesis of a distinct set of proteins called heat shock proteins (HSPs) (16, 19). Among the most strongly induced HSP families are the -crystallin-related, small HSPs, which are a group of proteins having molecular masses of 15 to 30 kDa (3, 11, 30). Unlike other groups of HSPs such as HSP70 and HSP60, small HSPs are not highly conserved (3, 11, 30). Sequence similarity is limited only to their carboxy-terminal regions. Various functions for small HSPs have been suggested, including protection against heat shock (3, 11, 30). Recent in vitro studies with recombinant small HSPs suggest that the small HSPs act in vivo as a type of molecular chaperone (3, 4, 11, 12, 30).

Prokaryotic homologs of small HSPs have been identified in Bacillus subtilis (10), Bradyrhizobium japonicum (17), Clostridium acetobutylicum (24), Escherichia coli (2), Mycobacterium leprae (18), Mycobacterium tuberculosis (29), Stigmatella aurantiaca (7, 8), and Streptomyces albus (25). Small HSPs from prokaryotes such as C. acetobutylicum (24), E. coli (2), Streptomyces albus (25), and Stigmatella aurantiaca (8) have been shown to be induced by elevated temperatures, although a homolog from Bacillus subtilis, CotM, was not induced by heat shock (10). Developmentally regulated expression of small HSPs from Bacillus subtilis (10), C. acetobutylicum (24), and Stigmatella aurantiaca (7, 8) have been demonstrated.

Like other organisms, cyanobacteria, which are oxygenic photosynthetic bacteria, synthesize a diverse range of HSPs upon exposure to high temperatures (31). However, the regulatory mechanism for the expression of cyanobacterial HSPs and their physiological functions remain poorly understood. Recently, an open reading frame (ORF), sll1514, which was designated hspA, was discovered during the genome sequencing project for Synechocystis sp. strain PCC 6803, a mesophilic cyanobacterium (13). The ORF has sequence homology with Stigmatella aurantiaca hspA, which encodes a small HSP homolog (8). This is the first indication of the presence of a small HSP in cyanobacteria. However, it is not known if the sll1514 ORF is actually expressed or not.

Synechococcus vulcanus, a thermophilic cyanobacterium, accumulates a 16-kDa protein along with GroEL and GroES as the major heat shock-induced proteins when the cells are shifted from 50 to 63°C (5, 21). Recently, we purified the 16-kDa protein to an apparent homogeneity (21). Size-exclusion chromatography and nondenaturing gel electrophoresis demonstrated that the 16-kDa protein formed large oligomeric structures of 280 kDa. The small HSPs from eukaryotes and a prokaryote (i.e., M. tuberculosis) are found in larger-molecular-mass complexes between 200 and 800 kDa that are most likely composed solely of small HSP subunits (3, 4, 11, 30).

In the present article, we report the isolation, sequence analysis, and in vivo transcription analysis of the gene encoding the 16-kDa protein from Synechococcus vulcanus.

Organisms and culture conditions. The thermophilic unicellular cyanobacterium Synechococcus vulcanus (15) was grown at 50°C in a liquid medium used for another thermophilic cyanobacterium, Synechococcus sp. (14), under a light intensity of 50 µmol/m²/s. The culture was bubbled with air supplemented with 5% CO₂. E. coli JM109 [recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi (lac-proAB)/F' (traD36 proAB⁺ lacI^q lacZM15)] used in cloning experiments was grown in Luria-Bertani medium supplemented with ampicillin (50 µg/ml) when appropriate (22).

Cloning and sequencing of the gene encoding the 16-kDa protein from Synechococcus vulcanus. The following two degenerate oligonucleotide primers were used to amplify a part of the gene encoding the 16-kDa protein from Synechococcus vulcanus by PCR. One of the primers, 5'-GCIATICA(A/G)(C/A)GICA(A/G)ATGAA-3' was based on the amino-terminal sequence of the 16-kDa protein, AIQRQMN, and the other primer, 5'-CCIGGIA(A/G)(C/T)TCIAC(C/T)TT-3', was based on the internal amino acid sequence of the protein, KVELPG. These amino acid sequences (Fig. 1) were determined by Edman degradation by utilizing the purified 16-kDa protein from Synechococcus vulcanus (21). A single 140-bp product was amplified from the Synechococcus vulcanus genome (20 ng) after 30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 2 min at 72°C. The PCR product was subcloned in pGEM-T vector (Promega, Madison, Wis.) to yield pGEMSH. Then, DNA sequencing of the PCR product was done by the dideoxy termination method (23) with an AutoRead sequencing kit (Pharmacia, Uppsala, Sweden) and a DNA sequencer (DSQ-1; Shimadzu, Kyoto, Japan). The deduced amino acid sequence of the PCR product contained the amino-terminal and internal amino acid sequences of the 16-kDa protein. The PCR fragment was recovered by digesting pGEMSH with PstI and SphI and radiolabeled with [-³²P]dCTP (ICN Biochemicals, Costa Mesa, Calif.) by the multiprime labeling method as directed by the manufacturer (Amersham International plc, Little Chalfont, England), purified through a NICK column (Pharmacia), and then used as a specific probe for genomic library screening and the Southern and Northern blot analyses described below.

View larger version (53K): [in this window] [in a new window]   FIG. 1.   Nucleotide sequence of the hspA gene from Synechococcus vulcanus. Putative 10 and 35 promoter sequences (underlined), the transcriptional start site (indicated by a downward arrow), a potential ribosome binding site (indicated by boldface type), and an inverted repeat thought to represent an hspA transcriptional terminator (indicated by horizontal arrows below the sequences) are marked. Only relevant restriction sites are depicted above the DNA sequence. The deduced amino acid sequence of the gene is shown below the DNA sequence (single-letter code). The translation stop signal is marked by an asterisk below the codon. The underlined amino acid sequences correspond to the sequences determined from the purified 16-kDa protein from Synechococcus vulcanus (21).

Southern blot analysis. We determined the number of copies of hspA in the Synechococcus vulcanus genome by Southern blot analysis. Synechococcus vulcanus genomic DNA digested with several different restriction endonucleases was hybridized with the radiolabeled 140-bp PCR-generated fragment (data not shown). The probe hybridized to only one DNA fragment from each restriction endonuclease digest. Similar experiments were repeated with another probe prepared with a 0.6-kbp EcoRI fragment containing a 3' portion of the hspA coding region (Fig. 1) that includes sequence encoding the conserved consensus region I (30). Results with this probe corresponding to a higher conserved region together with the 140-bp PCR-generated fragment demonstrated that there is no other gene in the genome that is homologous to hspA.

Northern blot analysis. A 500-ml culture of exponentially growing Synechococcus vulcanus cells at 50°C whose optical density at 730 nm was approximately 0.5 was divided into three portions. Under the same aeration and light conditions, one portion was kept at 50°C and the others were shifted to a 63°C bath and incubated for 15 or 60 min. After heat treatment, cells harvested by centrifugation at 4°C were immediately frozen in liquid nitrogen. Total RNA was isolated by the hot-phenol extraction method described previously (1). Total RNAs (5 µg) were electrophoresed on a denaturing 1.5% (wt/vol) agarose gel containing 6.6% (wt/vol) formaldehyde, transferred to a BA-S 83 nitrocellulose membrane in 20× SSC (1× SSC is 0.15 M NaCl containing 0.015 M sodium citrate) by a capillary transfer method (22), and then cross-linked to the membrane by UV illumination (FUNA-UV-Linker; Funakoshi, Tokyo, Japan). Prehybridization with the membrane was performed in a solution containing 6× SSC-5× Denhardt's solution (22)-0.1% SDS for 1 h at 65°C, and then hybridization was done in a solution of the same constituents containing the denatured radiolabeled 140-bp PCR fragment and 100 µg of denatured salmon sperm DNA per ml at 65°C overnight. After hybridization, the membrane was washed once in 6× SSC at room temperature for 5 min and, thereafter, twice in 6× SSC at 65°C for 30 min. The size of each mRNA was determined by using an RNA ladder (Gibco-BRL, Gaithersburg, Md.). The hybridization signals were detected and quantified with a BAS1000 Mac bio-imaging analyzer (Fuji Film, Tokyo, Japan).

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Northern blot analysis of the hspA (A) and groEL1 (B) transcripts from Synechococcus vulcanus. Total RNA was isolated from cells grown at 50°C (0 min) or incubated for different time intervals (15 and 60 min) after cultures were shifted from 50 to 63°C. Five micrograms of RNA from each sample was electrophoresed on a 1.5% (wt/vol) agarose gel containing 6.6% (wt/vol) formaldehyde. The 140-bp PCR product complementary to a part of the hspA coding region (Fig. 1) was used as a specific probe of the hspA transcripts. After the analysis, the same membrane was used for reprobing with a radiolabeled 540-bp PCR product complementary to a part of the Synechococcus vulcanus groEL1 coding region (28) (B). The 0.7-kb signal originates from the previous hybridization with the hspA probe since the same membrane was used. The uppermost band (labeled G) is due to contaminating genomic DNA as judged from its size. It disappeared when the sample was treated with DNase I before being loaded onto the agarose gel (data not shown).

View larger version (42K): [in this window] [in a new window]   FIG. 3.   Effect of rifampin on the stability of hspA mRNA in Synechococcus vulcanus. A 500-ml culture of exponentially growing cells at 50°C was shifted to a 63°C bath after cells were harvested (0 time) (lane 1) and incubated for 15 min to induce hspA expression. Immediately after cells were harvested (lane 2), the culture was divided into four 80-ml portions. An 0.8-ml volume of rifampin (30 mg dissolved in 1 ml of 100% methanol) was added to each of two cultures, and 0.8 ml of 100% methanol was added to each of the other ones. Then, a pair of cultures with (lanes 3, 4, 5, and 6) or without (lanes 7, 8, 9, and 10) rifampin was left at 63°C (lanes 3, 4, 7, and 8), and the other pair of cultures was shifted back to 50°C (lanes 5, 6, 9, and 10). Cells were harvested at 5 (lanes 3, 5, 7, and 9) or 10 (lanes 4, 6, 8, and 10) min after rifampin or methanol addition. Harvested cells were immediately frozen in liquid nitrogen, and total RNA was isolated by the hot-phenol extraction method as described in the text. A 7.5-µg amount of RNA from each sample was electrophoresed on a 1.5% (wt/vol) agarose gel containing 6.6% (wt/vol) formaldehyde. The 0.6-kbp EcoRI fragment containing the 3' region of hspA (see Fig. 1) was used as a specific probe of the hspA transcripts. Hybridization and detection of the hybridization signals were performed by the nonradioactive method (AlkPhos Direct) as directed by the manufacturer (Amersham International plc.).

Effect of rifampin on the stability of hspA mRNA. To examine whether increased transcript stability could contribute to heat shock induction of Synechococcus vulcanus hspA, the decay of hspA mRNA which accumulated during 15 min of heat shock was monitored, at 50 and 63°C, after inhibition of transcription by the addition of rifampin (Fig. 3). It appeared that Synechococcus vulcanus hspA mRNA was less stable at 50 than at 63°C (Fig. 3, compare lanes 3 and 4 with lanes 5 and 6). The half-life of Streptomyces albus hsp18 mRNA at 30°C was also shorter than that at 41°C (26). Interestingly, the stability of the mRNA was significantly increased by the addition of rifampin (Fig. 3, compare lanes 3 and 4 with lanes 7 and 8). It suggests that expression of an unknown gene(s) affects the stability of hspA mRNA.

The transcriptional initiation site of hspA in Synechococcus vulcanus. To determine the 5' end of the hspA transcript, an oligonucleotide primer, 5'-GGTTCCCAACGAACGAGTGC-3', complementary to the 5' terminus of the hspA coding strand sequence (Fig. 1, nucleotides 433 to 452) was radiolabeled with [-³²P]dATP (ICN Biochemicals) by using T4 polynucleotide kinase. Ten microliters (20 µg) of total RNA isolated from heat-shocked or non-heat-shocked cells, 4 µl of fivefold-concentrated buffer, which was supplied with the reverse transcriptase described below, and 2 µl (4 pmol) of the labeled primer were mixed together, heated at 65°C for 10 min, and then incubated at room temperature for 1 to 2 h. Two microliters of 0.1 M dithiothreitol and 1 µl of 20 mM deoxynucleoside triphosphates were added to the annealed primer-template mixture, and the mixture was incubated at 42°C for 2 min. Primer extension was carried out by the addition of 1 µl (200 U) of SuperScript II RNase H reverse transcriptase (Gibco-BRL) and subsequent incubation at 42°C for 1 h. The extended products were precipitated with ethanol and resuspended in 4 µl of 10 mM Tris-HCl (pH 8.0)-1 mM EDTA-6 µl of formamide loading buffer (80% formamide, 10 mM EDTA [pH 8.0], 1 mg of xylenecyanol per ml, and 1 mg of bromophenol blue per ml). After heat denaturation, a 5-µl sample was loaded onto a 6% polyacrylamide-7 M urea sequencing gel for electrophoresis (Fig. 4). Products of dideoxynucleotide sequencing reactions performed with the same primer and the cloned 3.0-kbp XbaI fragment as a template were run in parallel to allow determination of the end points of the primer extension products.

View larger version (53K): [in this window] [in a new window]   FIG. 4.   Mapping of the 5' end of hspA mRNA by primer extension analysis. Primer extension reactions were performed with the radiolabeled oligonucleotide primer complementary to the 5'-end coding region of hspA, and total RNA (20 µg) which was isolated from Synechococcus vulcanus cells before (lane 1) and after (lane 2) shifting from 50 to 63°C for 15 min. T, G, C, and A indicate the dideoxy sequencing ladder obtained with the same primer and the pBS II KS (+) derivative described in the text as a template. The potential transcription start site is indicated by an arrow.

Concluding remarks. In the present study, we have cloned and sequenced hspA, a gene encoding the 16-kDa protein. The deduced amino acid sequence of the gene product showed significant homology to the sequence of other small HSPs from prokaryotes and eukaryotes. Thus, the sequence comparison as well as the structural analysis (21) confirmed that the 16-kDa protein belongs to the family of small HSPs.

Nucleotide sequence accession number. The nucleotide sequence reported here has been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB002666.