Expression analysis of multiple dnaK genes in the cyanobacterium Synechococcus elongatus PCC 7942

Abstract

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.

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 β-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).

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.

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).

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.

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 β-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.

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) β-Galactosidase activity measured as in Fig. 5.