Abstract
The changes in the expression of sigma factor genes during dehydration in terrestrial Nostoc HK-01 and aquatic Anabaena PCC 7120 were determined. The expression of the sigJ gene in terrestrial Nostoc HK-01, which is homologous to sigJ (alr0277) in aquatic Anabaena PCC 7120, was significantly induced in the mid-stage of dehydration. We constructed a higher-expressing transformant of the sigJ gene (HE0277) in Anabaena PCC 7120, and the transformant acquired desiccation tolerance. The results of Anabaena oligonucleotide microarray experiments showed that a comparatively large number of genes relating to polysaccharide biosynthesis were upregulated in the HE0277 cells. The extracellular polysaccharide released into the culture medium of the HE0277 cells was as much as 3.2-fold more than that released by the control cells. This strongly suggests that the group 3 sigma factor gene sigJ is fundamental and conducive to desiccation tolerance in these cyanobacteria.
Key words: desiccation tolerance, sigma factor, exopolysaccharide, cyanobacteria
1. Introduction
Cyanobacteria grow in diverse habitats on earth, ranging from the tropics to polar regions.1 Terrestrial cyanobacteria are subjected to repeated cycles of dehydration, desiccation, and rehydration, and can survive under desiccated conditions for long periods. It has been reported that a terrestrial cyanobacterium Nostoc commune, which was stored in a desiccated state for more than 100 years, retained its ability to grow.2,3 Nostoc commune cells actively deactivate their photosynthetic systems on sensing water loss,4 and can recover the activities of photosystems I and II readily after rehydration.5 Nostoc commune cells produce a large amount of extracellular polysaccharide (EPS), which contributes to the stabilization of the cells during desiccation.6 Light intensity and the availability of combined nitrogen affect the synthesis of soluble EPSs in Nostoc strains.7,8 Despite many studies of the mechanism of desiccation tolerance, the molecular responses of cells to dehydration, desiccation, and rehydration remain unclear.
A sigma factor for RNA polymerase is capable of specific promoter recognition and the efficient initiation of transcription. Environmental or developmental signals change gene expression by inducing sigma factors in diverse bacterial species.9 There are two basic families of eubacterial sigma factors based on sequence similarities: the σ70 and σ54 families. The σ70 family is divided into three groups. Group 1 contains the primary sigma factors, which are essential for cell growth. Group 2 contains the sigma factors that are not essential for cell growth, but are similar to the primary sigma factors in the amino acid sequences of their DNA-binding regions. Group 3 includes the sigma factors that are only slightly homologous to groups 1 and 2 in their amino acid sequences. They regulate the transcription of specific regulons expressed in response to changes in environmental or developmental conditions. No sigma factor of the σ54 family has been found in the cyanobacteria. Twelve putative sigma factor genes exist in the genome and plasmids in Anabaena sp. PCC 7120.10 However, there have been few reports of the corresponding functions of the sigma factors. sigA, which encodes a primary sigma factor, was isolated and characterized under conditions of nitrogen limitation.11 The transcripts of the sigB and sigC genes, which encode group 2 sigma factors, are detectable under nitrogen-limited conditions.12 However, neither gene is essential for nitrogen fixation or heterocyst differentiation.13 The functions of the group 3 sigma factors of the PCC 7120 strain have not been reported at all.
We recently reported a comparative analysis of the differences in gene expression during dehydration between the terrestrial desiccation-tolerant cyanobacterium Nostoc sp. HK-01 and the aquatic cyanobacterium Anabaena PCC 7120.14 The gene expression changes were transient in Anabaena PCC 7120, whereas in Nostoc sp. HK-01, they were maintained or increased until the wet weight decreased to 10% of that before drying. Therefore, we inferred that sigma factors play important roles in gene expression during dehydration, because sigma factors play key roles in the initiation of transcription and gene expression. Here, we analysed the changes in the expression levels of the sigma factor genes in both Nostoc sp. HK-01 and Anabaena PCC 7120 during dehydration. We present significant evidence that sigJ gene plays an important role in desiccation tolerance.
2. Materials and methods
2.1. Bacterial strains and culture conditions
Transformant cells of Anabaena PCC 7120 were grown in BG11 medium15 at 30 °C under continuous illumination at 30 µE m−2 s−1 provided by fluorescent lamps. Nostoc sp. HK-01 was grown at 30 °C in WK medium16 with 5 mM N-tris-(hydroxymethyl)methyl-2-aminoethanesulphonic acid (TES)–NaOH (pH 8.0) under continuous illumination at 30 µE m−2 s−1 provided by fluorescent lamps. Liquid cultures were bubbled with air containing 1% CO2.
2.2. Drought stress conditions
A 10-mL portion of cell culture was harvested by filtration on a filter paper at a chlorophyll concentration of 15 µg chlorophyll/mL and dried on a plastic dish in an incubator under light (30 µE m−2 s−1) at 30 °C and 30–40% relative humidity. The cells were cryopreserved when the wet weight had decreased to 50, 30, and 10%, and cells harvested immediately before drying (100% wet weight) were used as the control.
2.3. Total RNA preparation
Total RNA was isolated from prepared cells by the hot-phenol method.17 Crude total RNA was treated with 0.1 U/μL DNase I (Takara Bio, Shiga, Japan) at 37 °C for 3 h. After phenol–chloroform–isoamyl alcohol extraction and ethanol precipitation, the total RNA was suspended in RNase-free ultrapure water.
2.4. Real-time quantitative reverse transcriptase–polymerase chain reaction
Aliquots (1 µg) of total RNA were reverse-transcribed with an RNA PCR Kit (Takara Bio). An aliquot (1/20) of the reaction mixture was taken for real-time quantitative polymerase chain reaction (PCR) with a set of gene-specific primers (Table 1). Reactions were performed with a DNA Engine Opticon® 2 System (Bio-Rad, CA, USA) using SYBR® Premix Ex Taq™ (Takara Bio) in the presence of 250 nM primers under the following conditions: 1 min at 95 °C, followed by 40 cycles of 5 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. The fluorescence intensity of the SYBR Green dye was measured after each amplification step. The relative ratios were presented as the means of data from duplicate or triplicate experiments. The primers used were designed using Primer3.18 Each primer for the sigma factors of Anabaena PCC 7120 was designed based on information from CyanoBase. Each specific primer for the sigma factors of Nostoc sp. HK-01 was designed on the basis of partial sequencing data using PCR products amplified with primers based on the conserved DNA sequence of the corresponding gene among Anabaena PCC 7120, Anabaena variabilis ATCC 29413, and Nostoc punctiforme ATCC 29133. Each datum for Nostoc sp. HK-01 was normalized to the value for 16S rRNA. Because the amount of transcript product of the 16S rRNA gene decreases as dehydration progresses in Anabaena PCC 7120, the quantitative determination was based on the amount of total RNA measured with a spectrophotometer. The primers are listed in Table 1. Product identification was confirmed by melting-curve analysis.
Table 1.
Primers used in real-time quantitative RT–PCR
| 16S rRNA | 5′-TGTAGCGGTGAAATGCGTAG-3′ | 5′-TTCACACTTGCGTGCGTACT-3′ |
|---|---|---|
| Anabaena PCC 7120 | ||
| all5263 (sigA) | 5′-GGTAGCCTTGGTTTGATTCG-3′ | 5′-CCATGCGAGTAGCGATTTCT-3′ |
| alr3800 (sigB2) | 5′-CACCCACACAGAAGACACAAA-3′ | 5′-CGTGGTCAGCCCACTCTG-3′ |
| all1692 (sigC) | 5′-CAGATGCCGCCTACAATACC-3′ | 5′-TTCGTCCCGTCCTAACAAAC-3′ |
| alr3810 (sigD) | 5′-CCACAACGTTGCAAGAAATG-3′ | 5′-CGCAAGTTGGCTTCTACCAT-3′ |
| alr4249 (sigE) | 5′-CGCTAATGATGCCAGAACCA-3′ | 5′-CCGACACGATGGTTTAAAGA-3′ |
| all3853 (sigF) | 5′-CGCTACTTGGCGATACAACA-3′ | 5′-CAATTCTCCCAGGCTAGTGG-3′ |
| alr3280 (sigG) | 5′-GTTGATGCGAGGTGTCCAG-3′ | 5′-CACGCGAATCCAAACTTCTT-3′ |
| alr0277 (sigJ) | 5′-AACTAAGTTGGCTGCCCAAA-3′ | 5′-TAGCAGCATCTTTGCGAGAA-3′ |
| Nostoc HK-01 | ||
| sigA | 5′-TTGCTTCTCGTTGATGATGG-3′ | 5′-CGCGCTAATTCGATTTCTTC-3′ |
| sigB2 | 5′-AACCCGTGAGCAAGAAATTG-3′ | 5′-TCCGAGTGTTCCTTCTTGGA-3′ |
| sigC | 5′-GCCACCGTCCATCTTTAGAA-3′ | 5′-GAAACCACAAGACGCAGGTT-3′ |
| sigD | 5′-AGATCGGCCGTGTACCACT-3′ | 5′-CATTCTCCGTTTCGCAATTT-3′ |
| sigE | 5′-GCTCAACGCGAACTTAAACA-3′ | 5′-TCTTTACCGACACGGTGGTT-3′ |
| sigF | 5′-TAAAGAATCTCGCTCGGAAA-3′ | 5′-AAGGAACTGAAAGCGTGTCC-3′ |
| sigG | 5′-TAGGATTGCGTCCAGATCGT-3′ | 5′-CCGATACACTCGAATCCACA-3′ |
| sigJ | 5′-GCGTTTAGTTCCTTTGCTGTG-3′ | 5′-CTGAATCTTTGGGAGGACGA-3′ |
2.5. Construction of transformants
The plasmid used for transformation in this study was based on pRL490, a stable shuttle vector in Anabaena species derived from a hybrid of pBR322 and the plasmid pDU1.19–23 pRL490 contains the tac promoter derived from Escherichia coli, the luxAB genes derived from Vibrio fischeri downstream from the promoter, and a gene conferring resistance to neomycin. The luxAB genes were removed from the plasmid with BamHI restriction endonuclease. The resultant plasmid was named pRL490–luxAB. DNA fragments of the sigJ gene were amplified by PCR with the set of primers 5′-GGATCCATGGCAGCAAGTGAGTCC-3′ and 5′-GGATCCCTATGAACCAGTAGGCAT-3′ using the genomic DNA of Anabaena PCC 7120 as the template. The primer set was designed to allow the introduction of a BamHI restriction site. The PCR products were cloned into pGEM-T Easy (Promega) according to the manufacturer's instructions. Cloning of the DNA fragments was verified by DNA sequencing. The fragment digested with BamHI was cloned into the BamHI restriction site of pRL490–luxAB to construct pRHE0277. pRHE0277 was transferred by conjugation into Anabaena PCC 7120 according to Elhai and Wolk24 and the transformant HE0277 was selected for resistance to neomycin. The control transformant P490 was obtained by transferring pRL490–luxAB into Anabaena PCC 7120 as described above.
2.6. Test for recovery from desiccation
P490 and HE0277 cells grown in liquid culture to stationary phase (OD750 = 3.0 ± 0.3) were filtered on a cellulose acetate membrane filter (Toyo Roshi Co., Ltd., Tokyo, Japan), dried for 10 h under light (30 µE m−2 s−1) at 30 °C and 30–40% relative humidity in an incubator, and stored for 1 week in the dark in the incubator. These dried cells were soaked with BG11 medium for 10 min. After the cell densities were adjusted (OD750 = 1.0, 0.1, and 0.01), the cell solutions were spread on an agar plate with BG11 medium and incubated under continuous light (30 µE m−2 s−1) at 30 °C.
2.7. DNA microarray analysis
DNA microarray analysis was performed according to Ehira and Ohmori.25 The analysis was conducted using three sets of total RNA samples extracted independently from the cells of P490 and HE0277. The ratio of the transcript level of the HE0277 strain relative to that of P490 strain was calculated from three measurements. Open reading frames (ORFs) whose spots showed signal intensities of more than twice the average intensity were analysed.
2.8. Extraction of EPSs
Cells (OD750 = 3.3 ± 0.3) and culture media were separated by centrifugation. This separation step was repeated three times. The harvested cells were resuspended in 0.05% sodium tetrahydridoborate solution and boiled at 100 °C for 1 h. The polysaccharide contents of the culture media and cell surfaces of the P490 and HE0277 cells were determined by the phenol–sulphuric acid method, using glucose as the standard.26
3. Results
3.1. Sigma factors responsive to drought stress
To determine the relationships of the sigma factors of Anabaena sp. PCC 7120 to those of other cyanobacteria, we aligned the sigma factor amino acid sequences from Anabaena sp. PCC 7120, Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, and A. variabilis (ATCC 29413) and constructed a phylogenetic tree (Fig. 1). Anabaena sp. PCC 7120 has one representative of the group 1 sigma factors, SigA; group 2 sigma factors, SigC, SigD, and SigE; and group 3 sigma factors, SigF, SigG, and SigI. The multiple paralogues of SigB, a group 2 sigma factor, have been classified as SigB (All7615), SigB2 (Alr3800), SigB3 (All7608), and SigB4 (All7179). sigB2 is encoded on the chromosome, whereas sigB, sigB3, and sigB4 are encoded on plasmids. A novel type of group 3 sigma factor was isolated and designated SigJ. Anabaena sp. PCC 7120 possesses a SigJ-type sigma factor, Alr0277, but not SigH-type sigma factor.
Figure 1.
Phylogenetic analysis of the sigma factors of Anabaena sp. PCC 7120, A. variabilis ATCC 29413, Synechocystis sp. PCC 6803, and Synechococcus sp. PCC 7942 with these full-length amino acid query sequences. The principal sigma factor (RpoD) sequence of E. coli is included as an outgroup. Multiple alignment analysis was performed with ClustalW software, and the phylogenetic tree was drawn with TreeView software. Numbers at the nodes are bootstrap values calculated with 1000 replications. Designations and accession numbers for amino acid sequences of the sigma factors are as follows: E. coli RpoD (P00579) from GenBank; the sigma factors of Anabaena sp. PCC 7120 from CyanoBase (http://www.kazusa.or.jp/cyano/); the sigma factors of A. variabilis ATCC 29413 from CYORF (http://cyano.genome.jp/) and JGI; the sigma factors of Synechocystis sp. PCC 6803 from CyanoBase; and the sigma factors of Synechococcus sp. PCC 7942 from CYORF.
We analysed the expression of the sigma factor genes during dehydration in Anabaena PCC 7120 (hereafter Anabaena) and Nostoc sp. HK-01 (hereafter Nostoc) using a real-time quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) method (Table 2). Anabaena has 12 putative genes for sigma factors on its chromosome and plasmids: nine genes exist on the chromosome and three genes on plasmids. We analysed the expression of the sigA (all5263), sigB2 (alr3800), sigC (all1692), sigD (alr3810), sigE (alr4249), sigF (all3853), sigG (alr3280), and sigJ (alr0277) genes on the chromosome of Anabaena and that of the corresponding genes of Nostoc. No expression of sigB (all7615), sigB3 (all7608), or sigB4 (all7179) was detected before or during dehydration in either species. sigI (all2193), which encodes a probable sigma factor, was not analysed because it is not a common gene in filamentous cyanobacteria. No orthologue exists in the genome of A. variabilis ATCC 29413 or N. punctiforme ATCC 29133. The transcript levels of sigA decreased as dehydration progressed in both Anabaena and Nostoc. However, the transcript level decreased to only about 53% in Anabaena, whereas it decreased to 13% in Nostoc when the wet weight had decreased to 50% of that before dehydration (Table 2).
Table 2.
Alteration of sigma factor gene expression during dehydration evaluated by real-time quantitative RT–PCR
| Wet weight change | 50% | 30% | 10% | |||
|---|---|---|---|---|---|---|
| Ratio (50%/100%) | SD | Ratio (30%/100%) | SD | Ratio (10%/100%) | SD | |
| N. HK-01 sigA | 0.13 | ±0.04 | 0.20 | ±0.03 | 0.12 | ±0.01 |
| N. HK-01 sigB2 | 1.17 | ±0.11 | 2.92 | ±0.04 | 2.17 | ±0.27 |
| N. HK-01 sigC | 1.27 | ±0.12 | 1.95 | ±0.36 | 1.90 | ±0.10 |
| N. HK-01 sigD | 0.94 | ±0.01 | 2.17 | ±0.15 | 1.20 | ±0.29 |
| N. HK-01 sigE | 1.27 | ±0.16 | 2.68 | ±0.12 | 1.29 | ±0.02 |
| N. HK-01 sigF | 0.16 | ±0.03 | 0.17 | ±0.01 | 0.03 | ±0.01 |
| N. HK-01 sigG | 1.16 | ±0.08 | 2.30 | ±0.16 | 2.18 | ±0.04 |
| N. HK-01 sigJ | 2.47 | ±0.22 | 1.43 | ±0.23 | 0.80 | ±0.03 |
| A. PCC 7120 sigA | 0.53 | ±0.10 | 0.10 | ±0.03 | 0.07 | ±0.02 |
| A. PCC 7120 sigB2 | 1.83 | ±0.01 | 0.55 | ±0.10 | 0.52 | ±0.02 |
| A. PCC 7120 sigC | 0.51 | ±0.004 | 0.10 | ±0.0001 | 0.09 | ±0.02 |
| A. PCC 7120 sigD | 0.57 | ±0.04 | 0.12 | ±0.02 | 0.08 | ±0.002 |
| A. PCC 7120 sigE | 1.63 | ±0.07 | 0.50 | ±0.01 | 0.34 | ±0.01 |
| A. PCC 7120 sigF | 1.17 | ±0.21 | 0.23 | ±0.01 | 0.18 | ±0.04 |
| A. PCC 7120 sigG | 1.28 | ±0.09 | 0.16 | ±0.02 | 0.16 | ±0.02 |
| A. PCC 7120 sigJ | 1.02 | ±0.42 | 0.13 | ±0.01 | 0.13 | ±0.02 |
The ratio values were obtained by calculating the ratio of the transcript level at each wet weight to the transcript level at the wet weight (100%) immediately before drying. Each datum is an average (±standard deviation) of three independent experiments.
The transcript levels of several group 2 sigma factors decreased totally as dehydration progressed in Anabaena. The transcript levels of sigC and sigD continued to decrease during dehydration. Transcript levels of sigB2 and sigE finally decreased, although they increased transiently at a wet weight of 50%. Conversely, the transcript levels of group 2 sigma factors increased significantly in Nostoc. The transcript levels of sigB2 and sigC were maintained at approximately twofold higher than the control level until the late stage of dehydration. The transcript levels of sigD and sigE were significantly increased, although transiently, at a wet weight of 30%.
In Anabaena, the transcript levels of several group 3 sigma factors did not change until the mid-stage of dehydration and decreased markedly in the late stage of dehydration. In Nostoc, the transcripts of sigF were strongly repressed until the mid-stage of dehydration. The transcript level of sigG was maintained at a level over twofold higher than that of the control in the late stage of dehydration. The transcript level of sigJ increased significantly in the mid-stage of dehydration, and then decreased gradually in the late stage of dehydration. It is noteworthy that among the sigma factor genes, the expression of sigJ was upregulated first when the cells were exposed to drought stress.
3.2. Comparison of the transcript levels of each sigma factor in Anabaena and Nostoc during dehydration
The relative ratios of the transcript levels for each sigma factor in Anabaena and Nostoc were determined during dehydration using real-time quantitative RT–PCR (Table 3). The ratios for sigC, sigE, and sigG transcripts in Nostoc to those in Anabaena were approximately 100-fold, 10-fold, and 30-fold, respectively, at the late stage of dehydration. The relative ratio for sigJ was approximately 120-fold, even before drying, and the maximum ratio was approximately 1550-fold at a wet weight of 30%. The intensive expression of the sigJ gene in Nostoc should positively influence desiccation tolerance.
Table 3.
Relative ratios of the transcript level of each sigma factor gene at each wet weight in both Anabaena sp. PCC 7120 and Nostoc sp. HK-01
| Wet weight change | 100% | 50% | 30% | 10% |
|---|---|---|---|---|
| Relative ratio | ||||
| N. sigA/A. sigA | 1.79 | 0.64 | 5.34 | 3.75 |
| N. sigB2/A. sigB2 | 0.40 | 0.23 | 2.76 | 1.52 |
| N. sigC/A. sigC | 6.19 | 14.74 | 103.25 | 95.54 |
| N. sigD/A. sigD | 0.15 | 0.26 | 2.13 | 2.66 |
| N. sigE/A. sigE | 2.69 | 2.29 | 13.44 | 10.77 |
| N. sigF/A. sigF | 58.8a | 5.51a | 43.05a | 5.82a |
| N. sigG/A. sigG | 2.86 | 2.59 | 34.18 | 33.31 |
| N. sigJ/A. sigJ | 123.30 | 411.86 | 1548.87 | 869.47 |
The value was calculated as the ratio of the transcript level of each sigma factor gene in Nostoc sp. HK-01 to its transcript level in Anabaena sp. PCC 7120. Relative ratios are the means of data from two independent experiments.
aTranscript levels were very low in both species and the error margins of the calculated values were large.
3.3. Recovery from desiccation of P490 and HE0277 cells
A transformation method for cells of Nostoc sp. HK-01 has not been established. Therefore, we produced clones of the Anabaena cell strain HE0277, which strongly express the Anabaena sigJ gene, and tested their recovery from desiccation based on the facts that the expression of sigJ was the first among the Nostoc sigma factor genes to be upregulated by drought stress and that sigJ transcripts exist more abundantly in Nostoc than in Anabaena. The cell strain transformed with a non-insertional pRL490–luxAB vector was used as the control strain, P490. As the result of Western blotting with anti-SigJ sera, the translation product level of sigJ in HE0277 cells was fourfold higher than the control level (data not shown). The HE0277 cells were better able to recover from the desiccated state than were the control cells (Fig. 2). Therefore, the numbers of colonies formed, as an indicator of recovery from the desiccated state, were determined for both strains when spread on a BG11 agar plate, after cell density was adjusted (OD750 = 0.01). From the results of eight measurements, the number of colonies formed by the HE0277 strain was 3.1-fold higher than that of the P490 strain (data not shown). Thus, the HE0277 cells were more desiccation-tolerant than the P490 cells.
Figure 2.
Recovery from desiccation of P490 and HE0277 cells. P490 and HE0277 cells were dried as described in Section 2. After the cell densities were adjusted (OD750 = 1.0, 0.1, and 0.01) using BG11 liquid medium, the cell solutions were spread on agar plates with BG11 medium and incubated for 7 days under continuous light (30 µE m−2 s−1) at 30 °C.
3.4. Effects of higher expression of the sigJ gene in Anabaena
The effects of the elevated expression of the sigJ gene were determined with an Anabaena oligonucleotide microarray. We found that the expression of 112 genes, including sigJ, was upregulated and that the expression of 42 genes was downregulated in HE0277 cells relative to that of P490 cells. Among the upregulated genes, 5% or more of the entire number of genes were included in the categories ‘cell envelope’, ‘photosynthesis and respiration’, ‘transcription and regulatory functions’, and ‘other categories’ (Table 4). The genes encoding probable glycosyl transferase or glucosyl transferase in ‘other categories’ accounted for 14.4% of the total number of upregulated genes. There were no particular features of the downregulated genes (Table 4 and Supplementary Table 1).
Table 4.
Functional categories of genes differentially expressed in P490 and HE0277 cell strains
| Categoriesa | Number of upregulated genes | Number of downregulated genes |
|---|---|---|
| Amino acid biosynthesis | 0 | 2 |
| Biosynthesis of cofactors, prosthetic groups, and carriers | 0 | 3 |
| Cell envelope | 6 | 0 |
| Cellular processes | 1 | 0 |
| Central intermediary metabolism | 3 | 0 |
| Energy metabolism | 1 | 1 |
| Fatty acid, phospholipid, and sterol metabolism | 0 | 0 |
| Photosynthesis and respiration | 6 | 2 |
| Nitrogen assimilation and fixation | 0 | 0 |
| Purines, pyrimidines, nucleosides, and nucleotides | 0 | 0 |
| Transcription and regulatory functions | 9 | 1 |
| DNA replication, recombination, and repair | 1 | 1 |
| Translation | 2 | 1 |
| Transport and binding proteins | 2 | 2 |
| Other categories (glucosyl transferase or glycosyl transferase) | 21 (16) | 6 (1) |
| Hypothetical and unknown | 59 | 23 |
| Total | 111 | 42 |
aGene categories were defined according to the CyanoBase database (http://www.kazusa.or.jp/cyano/cyano.html). sigJ is excluded from this table.
The details of the upregulated genes in HE0277 cells compared with those in the P490 cells are shown in Table 5. There are two gene clusters upregulated in the HE0277 cells. First, the gene cluster encoding ORFs all4003–asl3998 was uniformly upregulated. The isiA (all4001) encodes the chlorophyll-a-binding protein CP43′. isiB (alr2405), which encodes flavodoxin, an electron carrier that can substitute for iron-rich soluble ferredoxin, was also upregulated.27 Second, the expression of the gene cluster from alr3057 to alr3074 was uniformly upregulated in HE0277 cells. Because the upregulated expression of alr3074 was not statistically significant, it is not included in Table 5. However, the fold value (HE0277/P490) was 1.79 ± 0.27, indicating a tendency to upregulation. This region contains 18 ORFs and is 22.1 kbp long. According to CyanoBase, many genes relating to polysaccharide biosynthesis are included in this cluster.
Table 5.
Effects of higher expression of sigJ gene in Anabaena sp. PCC 7120
| ORFa | Producta | Fold (HE0277/P490)b | SD |
|---|---|---|---|
| all0394 | Hypothetical protein | 2.58 | ±2.41 |
| all0596 | Unknown protein | 2.10 | ±0.06 |
| all0721 | Unknown protein | 2.48 | ±1.61 |
| all0726 | Unknown protein | 3.61 | ±1.24 |
| all1009 | Unknown protein | 2.05 | ±0.50 |
| all1475 | Hypothetical protein | 2.01 | ±1.41 |
| all1708 | Unknown protein | 3.08 | ±0.02 |
| all1943 | Unknown protein | 2.13 | ±1.92 |
| all2200 | Unknown protein | 2.35 | ±2.13 |
| all2238 | Unknown protein | 2.42 | ±2.33 |
| all2290 | Similar to polysaccharide biosynthesis export protein | 2.43 | ±0.09 |
| all2573 | Unknown protein | 2.98 | ±1.82 |
| all2642 | Multifunctional peptide synthetase | 2.45 | ±2.15 |
| all2649 | Probable non-ribosomal peptide synthetase | 2.03 | ±1.51 |
| all2703 | Hypothetical protein | 2.24 | ±2.10 |
| all2770 | Dolichyl-phosphate-mannose synthase | 2.32 | ±0.17 |
| all2961 | Similar to reverse transcriptase | 2.10 | ±1.88 |
| all3317 | Unknown protein | 2.04 | ±0.22 |
| all3531 | Hypothetical protein | 2.13 | ±1.93 |
| all3743 | Transcriptional regulator | 2.44 | ±0.75 |
| all3999 | Unknown protein | 4.60 | ±1.71 |
| all4000 | Photosystem II CP43 protein PsbC homologue | 3.71 | ±0.88 |
| all4001 | Photosystem II chlorophyll-a-binding protein IsiA | 2.06 | ±0.18 |
| all4002 | Photosystem II CP43 protein PsbC homologue | 2.27 | ±1.52 |
| all4003 | Photosystem II CP43 protein PsbC homologue | 2.07 | ±0.14 |
| all4342 | Mannose-6-phosphate isomerase | 2.77 | ±0.53 |
| all4375 | Unknown protein | 2.01 | ±0.48 |
| all4420 | Glucosyl transferase | 2.00 | ±0.26 |
| all4422 | UDP-N-acetyl-d-mannosamine transferase | 2.26 | ±0.88 |
| all4459 | Unknown protein | 3.18 | ±0.91 |
| all4719 | Similar to glucosyl transferase | 13.14 | ±0.26 |
| all5091 | Unknown protein | 2.05 | ±1.45 |
| all5113 | Unknown protein | 2.07 | ±1.01 |
| all5221 | Unknown protein | 3.46 | ±0.23 |
| all5244 | Unknown protein | 2.46 | ±0.38 |
| all5245 | Unknown protein | 2.88 | ±0.28 |
| all5246 | Unknown protein | 3.27 | ±2.34 |
| all5247 | Unknown protein | 14.18 | ±3.95 |
| all5337 | Similar to TRK system potassium uptake protein | 2.43 | ±0.56 |
| alr0074 | Putative glycosyl transferase | 2.33 | ±0.23 |
| alr0242 | Hypothetical protein | 6.33 | ±0.84 |
| alr0248 | Unknown protein | 2.06 | ±0.09 |
| alr0277 | Group 3 sigma factor, SigJ | 144.17 | ±30.69 |
| alr0280 | Ribonuclease III | 2.60 | ±0.42 |
| alr0369 | Unknown protein | 5.56 | ±0.67 |
| alr0370 | Unknown protein | 10.21 | ±2.18 |
| alr0429 | Two-component response regulator | 5.66 | ±0.41 |
| alr0430 | Probable short-chain dehydrogenase | 2.94 | ±0.70 |
| alr0564 | Hypothetical protein | 2.35 | ±1.02 |
| alr0655 | Hypothetical protein | 2.08 | ±1.57 |
| alr0692 | Similar to NifU protein | 2.77 | ±0.81 |
| alr1016 | Hypothetical protein | 2.41 | ±2.01 |
| alr1183 | Unknown protein | 2.76 | ±1.30 |
| alr1372 | Hypothetical protein | 2.27 | ±1.56 |
| alr1534 | Hypothetical protein | 2.09 | ±0.80 |
| alr2071 | Unknown protein | 2.13 | ±2.03 |
| alr2142 | Oxidoreductase | 2.09 | ±1.36 |
| alr2174 | Transcriptional regulator | 2.10 | ±0.38 |
| alr2256 | Unknown protein | 2.95 | ±1.51 |
| alr2310 | Similar to agmatinase | 2.15 | ±0.99 |
| alr2405 | Flavodoxin, IsiB | 3.36 | ±0.74 |
| alr2407 | Hypothetical protein | 2.38 | ±0.41 |
| alr2447 | DnaJ protein | 2.15 | ±2.09 |
| alr2522 | Unknown protein | 2.07 | ±0.37 |
| alr2539 | Branched-chain amino acid ABC transporter, ATP-binding protein | 2.61 | ±2.29 |
| alr3057 | Probable glycosyl transferase | 2.63 | ±0.34 |
| alr3058 | Probable glycosyl transferase | 2.50 | ±0.21 |
| alr3059 | Similar to polysaccharide export protein | 2.86 | ±0.21 |
| alr3060 | Unknown protein | 3.20 | ±0.47 |
| alr3061 | Similar to acetyl transferase | 3.34 | ±0.51 |
| alr3062 | Probable glycosyl transferase | 4.74 | ±0.01 |
| alr3063 | Probable glycosyl transferase | 3.61 | ±0.29 |
| alr3064 | Probable glycosyl transferase | 3.57 | ±1.30 |
| alr3065 | Probable polysaccharide biosynthesis protein | 2.47 | ±0.20 |
| alr3066 | Polysaccharide polymerization protein | 2.53 | ±0.69 |
| alr3067 | Probable glycosyl transferase | 2.37 | ±0.55 |
| alr3068 | Probable glycosyl transferase | 3.09 | ±0.65 |
| alr3069 | Probable glycosyl transferase | 2.06 | ±0.11 |
| alr3070 | Probable glycosyl transferase | 3.18 | ±0.34 |
| alr3071 | Probable glycosyl transferase | 3.09 | ±0.16 |
| alr3072 | Probable polysaccharide biosynthesis protein | 3.36 | ±0.19 |
| alr3073 | Probable glycosyl transferase | 2.95 | ±0.92 |
| alr3092 | Two-component hybrid sensor and regulator | 2.19 | ±1.58 |
| alr3485 | Unknown protein | 2.09 | ±0.63 |
| alr3507 | Hypothetical protein | 3.40 | ±0.79 |
| alr3547 | Two-component sensor histidine kinase | 2.02 | ±1.54 |
| alr3548 | Hypothetical protein | 2.18 | ±2.06 |
| alr3554 | Unknown protein | 4.48 | ±2.54 |
| alr3594 | Two-component response regulator | 2.10 | ±0.35 |
| alr3666 | Urease accessory protein D | 2.30 | ±1.75 |
| alr3775 | Unknown protein | 2.11 | ±1.33 |
| alr3816 | Unknown protein | 15.90 | ±2.44 |
| alr3817 | Unknown protein | 5.53 | ±0.94 |
| alr3997 | Serine/threonine kinase | 2.04 | ±0.25 |
| alr4057 | Similar to peptide synthetase | 2.49 | ±0.29 |
| alr4132 | Hypothetical protein | 2.44 | ±0.83 |
| alr4800 | Similar to anti-sigma factor antagonist | 2.65 | ±0.10 |
| alr4818 | Similar to Bpu10I restriction endonuclease beta subunit | 2.49 | ±2.23 |
| alr4823 | Similar to glucosyl-1-phosphate transferase | 3.00 | ±1.05 |
| alr4965 | Hypothetical protein | 4.32 | ±1.68 |
| alr5223 | Glycosyl transferase | 3.68 | ±0.04 |
| alr5224 | Hypothetical protein | 3.33 | ±0.77 |
| asl0597 | Hypothetical protein | 2.21 | ±0.00 |
| asl2078 | Unknown protein | 2.01 | ±1.59 |
| asl3998 | Hypothetical protein | 3.42 | ±0.61 |
| asl5041 | Unknown protein | 2.51 | ±2.00 |
| asr0431 | Unknown protein | 2.05 | ±0.85 |
| asr0941 | Photosystem II protein PsbX | 3.20 | ±2.16 |
| asr1185 | Unknown protein | 2.06 | ±1.26 |
| asr1734 | Unknown protein | 3.20 | ±1.57 |
| asr2206 | Hypothetical protein | 2.67 | ±1.02 |
| asr4951 | Unknown protein | 3.51 | ±2.05 |
The fold values that exceeded 2.0 were considered significantly different between the two strains.
Each datum is the average (±standard deviation) calculated from three independent experiments.
The gene clusters relevant to putative polysaccharide biosynthesis and the ORFs encoding glycosyl/glucosyl transferase are shaded in grey.
a‘ORF’ and ‘description’ are defined according to the CyanoBase database.
bThe fold value was calculated as the ratio of the signal intensity of each gene of HE0277 to that of P490.
3.5. Analysis of the EPSs of HE0277 and P490 cells
Because a proportion of the genes were categorized as ‘cell envelope’ encoding, and the number designated glycosyl/glucosyl transferases was comparatively large relative to the entire number of genes upregulated (approximately 20%), we considered that the amount of EPS was increased in HE0277 cells. Each EPS in the P490 and HE0277 cells was collected from the cell surface and liquid culture, and analysed as described in Section 2. Significant differences were detected in the amounts of polysaccharides released into the culture medium, although no difference was detected in the amounts of cell-surface polysaccharides (Table 6). The amount of polysaccharides released by HE0277 cells was as much as 3.2-fold higher than that released by P490 cells.
Table 6.
Comparative analysis of EPSs in P490 and HE0277 cell strains
| Cell strain | Carbohydrate production | |||
|---|---|---|---|---|
| Cell surface (mg L−1) | SD | Medium (mg L−1) | SD | |
| P490 | 864.42 | ±24.16 | 516.86 | ±84.83 |
| HE0277 | 778.65 | ±40.56 | 1651.69 | ±70.17 |
Each datum is an average (±standard deviation) of three independent experiments.
EPS, extracellular polysaccharide.
4. Discussion
4.1. Role of sigma factors in drought stress
The expression of the sigA gene was repressed as dehydration progressed in both Anabaena and Nostoc. The sigA product regulates the transcription of a number of housekeeping genes and is principally involved in cell viability. Genes related to photosynthesis, carbon dioxide fixation, adenosine triphosphate (ATP) synthesis, transcription, and translation are downregulated under desiccation.28 The transcript level of sigA in Nostoc was strongly repressed at the mid-stage of dehydration (Table 2). It is necessary to stop cell processes during dehydration to conserve the energy needed to survive under desiccation. It is reasonable that Nostoc strictly controls the transcription level of the sigA gene to ensure survival.
Expression of the group 3 sigma factor gene, sigJ, was responsive to drought stress at the earliest stage of dehydration in Nostoc. The relative ratio of the number of transcripts of the sigJ gene in Nostoc to that in Anabaena was more than a hundred (Tables 2 and 3). The elevated expression of the sigJ gene must enable the cells of Nostoc to respond to drought stress in a timely manner. The higher expression of the sigJ gene in Anabaena resulted in the upregulation of genes related to polysaccharide biosynthesis (Tables 4 and 5), suggesting that SigJ governs the regulons of EPS biosynthesis. The expression of the gene cluster from alr3057 to alr3074 was upregulated in the HE0277 cells during dehydration, whereas it was downregulated in the desiccation-sensitive Anabaena.28 This gene cluster might be responsible for desiccation tolerance.
In the drying phase, cells avoid lysis from hyperosmotic-down shock and protect their DNA, RNA, proteins, and membranes from water stress, oxidative stress, and other stresses.29,30 It was considered that the upregulation of isiA and isiB expression in HE0277 cells, which are induced under conditions of light excess, iron-limited conditions, and oxidative conditions,31–34 is an example for desiccation tolerance. Genes regulated by the group 2 sigma factor RpoS of E. coli and the group 3 sigma factor SigB of Bacillus subtilis, which are general stress-responsive sigma factors, encode various functions that are involved in the prevention and repair of DNA damage, cell morphology, modulation of virulence genes, osmoprotection, thermotolerance, glycogen synthesis, membranes, and the cell envelope.9,35 However, sigJ gene is not the only sigma factor gene that responds to drought stress. Other group 2 or group 3 sigma factor genes of Nostoc are also responsive to drought stress in the late stage of dehydration (Tables 2 and 3). The function of the sigma factors in drought stress seems to differ from that of SigJ. Further analysis of the sigma factors is required to elucidate the desiccation tolerance of Nostoc species.
4.2. Significance of exopolysaccharides in desiccation tolerance
In the HE0277 strain, genes putatively concerning polysaccharide biosynthesis were upregulated, and at the same time, polysaccharides were excreted increasingly into the culture medium (Table 6). We investigated the monosaccharidic composition of the released polysaccharides produced by HE0277 and P490 cells. The dried polysaccharides were hydrolysed, neutralized, and desalted, and the monosaccharides were then quantified by high-performance anion-exchange chromatography, as described previously.36 The polysaccharides released by both HE0277 and P490 cells were composed of eight kinds of identified monosaccharides (galactose, glucose, fucose, rhamnose, arabinose, xylose, galacturonic acid, and glucuronic acid) and several unknown monosaccharides (data not shown). No difference in monosaccharidic composition was detected between the HE0277 and P490 strains. The amount of each monosaccharide in the polysaccharides released by HE0277 was two- to threefold greater than that released by P490. Many studies have been carried out to determine the function of EPSs in the capacity of some polysaccharide-producing cyanobacteria to overcome desiccation in the desert.37 It has been proposed that EPSs provide a repository for water, thereby acting as a buffer between cells and the atmosphere, and represent the key component of the mechanism used by cyanobacteria to tolerate desiccation.38 The EPSs of N. commune account for more than 60% of its dry weight.6 The polysaccharides, together with trehalose, may play a key role in dehydration tolerance.39 It has been reported that photosynthetic O2 evolution in EPS-depleted N. commune cells is sensitive to desiccation treatment, whereas that of normal N. commune cells is rapidly recovered after rehydration.40 Because HE0277 cells produced and released polysaccharides in increased amounts (Table 6), the polysaccharides must be a critical factor in desiccation tolerance. The group 3 sigma factor gene sigJ is fundamental to desiccation tolerance. This discovery will expedite our understanding of the mechanism of desiccation tolerance and EPS biosynthesis in cyanobacteria.
Acknowledgements
We have benefited from the excellent technical support of Mr Tsutomu Aohara and Dr Toshihisa Kotake, Saitama University, with the total sugar assay and the determination of monosaccharides. This work was supported by a Grant-in-Aid to the Japan Space Forum.
Supplementary Data
Supplementary data are available online at www.dnaresearch.oxfordjournals.org.
References
- 1.Potts M. Desiccation tolerance of prokaryotes. Microbiol. Rev. 1994;58:755–805. doi: 10.1128/mr.58.4.755-805.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Lipman C. B. The successful revival of Nostoc commune from a herbarium specimen eighty-seven years old. Bull. Torr. Bot. Club. 1941;68:664–666. [Google Scholar]
- 3.Cameron R. E. Species of Nostoc Vaucher occurring in the Sonoran Desert in Arizona. Trans. Am. Microsc. Soc. 1962;81:379–384. [Google Scholar]
- 4.Hirai M., Yamakawa R., Nishio J., et al. Deactivation of photosynthetic activities is triggered by loss of a small amount of water in a desiccation-tolerant cyanobacterium, Nostoc commune (Cyanobacteria) Plant Cell Physiol. 2004;45:872–878. doi: 10.1093/pcp/pch094. [DOI] [PubMed] [Google Scholar]
- 5.Satoh K., Hirai M., Yamakawa R., et al. Recovery of photosynthetic systems during rewetting is quite rapid in a terrestrial cyanobacterium, Nostoc commune. Plant Cell Physiol. 2002;43:170–176. doi: 10.1093/pcp/pcf020. [DOI] [PubMed] [Google Scholar]
- 6.Hill D. R., Keenan T. W., Helm R. F., Potts M., Crowe L. M., Crowe J. H. Extracellular polysaccharide of Nostoc commune (Cyanobacteria) inhibits fusion of membrane vesicles during desiccation. J. Appl. Phycol. 1997;9:237–248. [Google Scholar]
- 7.Otero A., Vincenzini M. Extracellular polysaccharide synthesis by Nostoc strains as affected by N source and light intensity. J. Biotechnol. 2003;102:143–152. doi: 10.1016/s0168-1656(03)00022-1. [DOI] [PubMed] [Google Scholar]
- 8.Otero A., Vincenzini M. Nostoc (Cyanophyceae) goes nude: extracellular polysaccharides serve as a sink for reducing power under unbalanced C/N metabolism. J. Phycol. 2004;40:74–81. [Google Scholar]
- 9.Wösten M. M. S. M. Eubacterial sigma-factors. FEMS Microbiol. Rev. 1998;22:127–150. doi: 10.1111/j.1574-6976.1998.tb00364.x. [DOI] [PubMed] [Google Scholar]
- 10.Kaneko T., Nakamura Y., Wolk C. P., et al. Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res. 2001;8:205–213. doi: 10.1093/dnares/8.5.205. 227–253. [DOI] [PubMed] [Google Scholar]
- 11.Brahamsha B., Haselkorn R. Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120. J. Bacteriol. 1991;173:2442–2450. doi: 10.1128/jb.173.8.2442-2450.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Brahamsha B., Haselkorn R. Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes. J. Bacteriol. 1992;174:7273–7282. doi: 10.1128/jb.174.22.7273-7282.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Khudyakov I. Y., Golden J. W. Identification and inactivation of three group 2 sigma factor genes in Anabaena sp. strain PCC 7120. J. Bacteriol. 2001;183:6667–6675. doi: 10.1128/JB.183.22.6667-6675.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Yoshimura H., Ikeuchi M., Ohmori M. Up-regulated gene expression during dehydration in a terrestrial cyanobacterium, Nostoc sp. strain HK-01. Microbes Environ. 2006;21:129–133. [Google Scholar]
- 15.Stanier R. Y., Kunisawa R., Mandel M., Cohen-Bazire G. Purification and properties of unicellular blue-green algae (order Chroococcales) Bacteriol. Rev. 1971;35:171–205. doi: 10.1128/br.35.2.171-205.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Katoh H., Shiga Y., Nakahira Y., Ohmori M. Isolation and characterization of a drought-tolerant cyanobacterium, Nostoc sp. HK-01. Microbes Environ. 2003;18:82–88. [Google Scholar]
- 17.Mohamed A., Jansson C. Influence of light on accumulation of photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803. Plant Mol. Biol. 1989;13:693–700. doi: 10.1007/BF00016024. [DOI] [PubMed] [Google Scholar]
- 18.Rozen S., Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 2000;132:365–386. doi: 10.1385/1-59259-192-2:365. [DOI] [PubMed] [Google Scholar]
- 19.Reaston J., van den Hondel C. A., van Arkel G. A., Stewart W. D. A physical map of plasmid pDU1 from the cyanobacterium Nostoc PCC 7524. Plasmid. 1982;7:101–104. doi: 10.1016/0147-619x(82)90032-4. [DOI] [PubMed] [Google Scholar]
- 20.Wolk C. P., Vonshak A., Kehoe P., Elhai J. Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc. Natl. Acad. Sci. USA. 1984;81:1561–1565. doi: 10.1073/pnas.81.5.1561. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Wolk C. P., Cai Y., Cardemil L., et al. Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen. J. Bacteriol. 1988;170:1239–1244. doi: 10.1128/jb.170.3.1239-1244.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Elhai J., Wolk C. P. Developmental regulation and spatial pattern of expression of the structural genes for nitrogenase in the cyanobacterium Anabaena. EMBO J. 1990;9:3379–3388. doi: 10.1002/j.1460-2075.1990.tb07539.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Elhai J. Strong and regulated promoters in the cyanobacterium Anabaena PCC 7120. FEMS Microbiol. Lett. 1993;114:179–184. doi: 10.1111/j.1574-6968.1993.tb06570.x. [DOI] [PubMed] [Google Scholar]
- 24.Elhai J., Wolk C. P. A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene. 1988;68:119–138. doi: 10.1016/0378-1119(88)90605-1. [DOI] [PubMed] [Google Scholar]
- 25.Ehira S., Ohmori M. NrrA, a nitrogen-responsive response regulator facilitates heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Mol. Microbiol. 2005;59:1692–1703. doi: 10.1111/j.1365-2958.2006.05049.x. [DOI] [PubMed] [Google Scholar]
- 26.Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A., Smith F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956;28:350–356. [Google Scholar]
- 27.Fromme P., Melkozernov A., Jordan P., Krauss N. Structure and function of photosystem I: interaction with its soluble electron carriers and external antenna systems. FEBS Lett. 2003;555:40–44. doi: 10.1016/s0014-5793(03)01124-4. [DOI] [PubMed] [Google Scholar]
- 28.Katoh H., Asthana R. K., Ohmori M. Gene expression in the cyanobacterium Anabaena sp. PCC 7120 under desiccation. Microb. Ecol. 2004;47:164–174. doi: 10.1007/s00248-003-1043-6. [DOI] [PubMed] [Google Scholar]
- 29.Potts M. Desiccation tolerance: a simple process? Trends Microbiol. 2001;9:553–559. doi: 10.1016/s0966-842x(01)02231-4. [DOI] [PubMed] [Google Scholar]
- 30.Billi D., Potts M. Life and death of dried prokaryotes. Res. Microbiol. 2002;153:7–12. doi: 10.1016/s0923-2508(01)01279-7. [DOI] [PubMed] [Google Scholar]
- 31.Leonhardt K., Straus N. A. Photosystem II genes isiA, psbDI and psbC in Anabaena sp. PCC 7120: cloning, sequencing and the transcriptional regulation in iron-stressed and iron-repleted cells. Plant Mol. Biol. 1994;24:63–73. doi: 10.1007/BF00040574. [DOI] [PubMed] [Google Scholar]
- 32.Park Y. I., Sandstrom S., Gustafsson P., Oquist G. Expression of the isiA gene is essential for the survival of the cyanobacterium Synechococcus sp. PCC 7942 by protecting photosystem II from excess light under iron limitation. Mol. Microbiol. 1999;32:123–129. doi: 10.1046/j.1365-2958.1999.01332.x. [DOI] [PubMed] [Google Scholar]
- 33.Xu W. L., Jeanjean R., Liu Y. D., Zhang C. C. pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp. PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA. FEBS Lett. 2003;553:179–182. doi: 10.1016/s0014-5793(03)01019-6. [DOI] [PubMed] [Google Scholar]
- 34.Havaux M., Guedeney G., Hagemann M., Yeremenko N., Matthijs H. C., Jeanjean R. The chlorophyll-binding protein IsiA is inducible by high light and protects the cyanobacterium Synechocystis PCC6803 from photooxidative stress. FEBS Lett. 2005;579:2289–2293. doi: 10.1016/j.febslet.2005.03.021. [DOI] [PubMed] [Google Scholar]
- 35.Hengge-Aronis R. Signal transduction and regulatory mechanisms involved in control of the σS (RpoS) subunit of RNA polymerase. Microbiol. Mol. Biol. Rev. 2002;66:373–395. doi: 10.1128/MMBR.66.3.373-395.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Ishikawa M., Kuroyama H., Takeuchi Y., Tsumuraya Y. Characterization of pectin methyltransferase from soybean hypocotyls. Planta. 2000;210:782–791. doi: 10.1007/s004250050680. [DOI] [PubMed] [Google Scholar]
- 37.De Philippis R., Vincenzini M. Exocellular polysaccharides from cyanobacteria and their possible applications. FEMS Microbiol. Rev. 1998;22:151–175. [Google Scholar]
- 38.Hill D. R., Peat A., Potts M. Biochemistry and structure of the glycan secreted by desiccation-tolerant Nostoc commune (Cyanobacteria) Protoplasma. 1994;182:126–148. [Google Scholar]
- 39.Higo A., Katoh H., Ohmori K., Ikeuchi M., Ohmori M. The role of a gene cluster for trehalose metabolism in dehydration tolerance of the filamentous cyanobacterium Anabaena sp. PCC 7120. Microbiology. 2005;152:979–987. doi: 10.1099/mic.0.28583-0. [DOI] [PubMed] [Google Scholar]
- 40.Tamaru Y., Takani Y., Yoshida T., Sakamoto T. Crucial role of extracellular polysaccharides in desiccation and freezing tolerance in the terrestrial cyanobacterium Nostoc commune. Appl. Environ. Microbiol. 2005;71:7327–7333. doi: 10.1128/AEM.71.11.7327-7333.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.Imamura S., Yoshihara S., Nakano S., et al. Purification, characterization, and gene expression of all sigma factors of RNA polymerase in a cyanobacterium. J. Mol. Biol. 2003;325:857–872. doi: 10.1016/s0022-2836(02)01242-1. [DOI] [PubMed] [Google Scholar]