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1 Department of Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-Ohkubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan
2 Department of Life Sciences (Biology), University of Tokyo, Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan
Correspondence
Masayuki Ohmori
ohmori{at}molbiol.saitama-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The microarray data for this paper have been deposited in the KEGG expression database; accession numbers are given in the text.
Seven supplementary tables with details of the up- and downregulated genes are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2001). It is widely believed that the accumulation of disaccharides, such as trehalose and sucrose, during the dehydration process confers desiccation tolerance. However, it has become increasingly clear that true desiccation tolerance is likely to involve several mechanisms working in concert. These include the scavenging of reactive oxygen species, the downregulation of metabolism, and the accumulation of certain amphiphilic solutes, proteins and polysaccharides (Oliver et al., 2001). The cellular responses to desiccation and rehydration are distinct from one another and are equally complex (Shaw et al., 2003; Singh et al., 2005). The strictly regulated resumption of cellular metabolic processes is required on rehydration (Potts, 1994; Scherer et al., 1984). Furthermore, some damage to DNA and membranes becomes manifest upon rehydration (Potts, 1994).
The N2-fixing filamentous cyanobacteria Nostoc commune can survive repeated cycles of desiccation and rehydration (Potts, 1994, 1999; Shaw et al., 2003). Desiccated N. commune rapidly recovers its photosynthetic activity after being soaked (Satoh et al., 2002; Scherer et al., 1984; Tamaru et al., 2005). An extracellular substance, such as exopolysaccharide (EPS), seems to function in desiccation tolerance (Helm et al., 2000; Hill et al., 1994a, b, 1997; Scherer & Potts, 1989; Shaw et al., 2003; Shirkey et al., 2000; Tamaru et al., 2005; Wright et al., 2005). Only a few studies of the intracellular mechanism of desiccation tolerance have been reported (Shirkey et al., 2000; Xie et al., 1995).
Anabaena sp. PCC 7120 (hereafter Anabaena PCC 7120), which is a close relative of the desiccation-tolerant Nostoc sp. HK-01 (Katoh et al., 2003), is also tolerant to desiccation, although only weakly (Higo et al., 2006; Katoh et al., 2004). The genome sequence of Anabaena PCC 7120 has been completely determined (Kaneko et al., 2001) and a genetic transformation system has been developed (Elhai et al., 1997). We have shown that Anabaena PCC 7120 accumulates large amounts of sucrose but only small amounts of trehalose (0.05–0.1 % of dry weight), although trehalose is important for desiccation tolerance (Higo et al., 2006). We have also previously reported microarray analysis of the genome-wide gene expression changes during dehydration (Higo et al., 2006; Katoh et al., 2004).
In this study, we performed DNA microarray analysis during rehydration following dehydration. We discuss the similarities and differences in transcriptional profiles during dehydration and rehydration. Whereas no genes encoding transcriptional regulators were specifically upregulated during dehydration, eight were specifically upregulated during rehydration. Of these, we focused on ancrpB and alr0618, encoding putative transcriptional regulators of the cAMP receptor protein (CRP) family.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) until late-exponential phase. MDM0 medium contains, per litre: 13 mg CaCl2 . 2H2O, 0.25 g MgSO4 . 7H2O, 0.1 g NaCl, 0.25 g K2HPO4, 20 mg FeSO4 . 7H2O and 1 ml trace elements A6 (Kratz & Myers, 1955). Liquid cultures were bubbled with air containing 1.0 % (v/v) CO2.
Dehydration stress was imposed on cells as described previously (Higo et al., 2006). A 30 ml portion of cell culture was filtered onto cellulose acetate filter paper (0.45 µm pore size, 47 mm diameter; Advantec) and dried for 24 h at 30 °C at 30–40 µmol photons m–2 s–1 in a Petri dish. After dehydration for 24 h, cells on the filter paper were rehydrated in 30 ml MDM0 and incubated as described above for the indicated times.
DNA microarray analysis.
RNA was extracted from cells as described previously (Katoh et al., 2004). DNA microarray analysis with an Anabaena oligonucleotide microarray (Ehira & Ohmori, 2006; Higo et al., 2006) (Sigma-Aldrich) was performed according to the method described previously (Higo et al., 2006) with some modifications.
A mixture of RNA and Anabaena primer mix (Ehira & Ohmori, 2006; Higo et al., 2006) (Sigma-Aldrich) in an 11 µl volume was heated to 90 °C for 5 min, and then gradually cooled to 42 °C for 20 min. Reverse transcription was performed at 42 °C for 90 min in a 20 µl volume containing 5 µg RNA, Anabaena primer mix (3.44 nM each ORF), 1x first-strand buffer (50 mM Tris/HCl, 75 mM KCl, 3 mM MgCl2, pH 8.3), 10 mM DTT, 250 µM each dATP, dGTP and dCTP, 200 µM dTTP, 50 µM Cy3-dUTP or Cy5-dUTP, 200 U SuperScript II Reverse Transcriptase (Invitrogen) and 20 U RNase inhibitor (Takara). The RNA in the reaction mixtures was hydrolysed by incubation at 70 °C after the addition of 10 µl 0.1 M NaOH, after which the mixtures were neutralized by the addition of 15 µl 1 M Tris/HCl (pH 7.5). Two reaction mixtures (one labelled with Cy3 and the other labelled with Cy5) were combined and purified with a QIAquick PCR Purification kit (Qiagen) to remove unincorporated fluorescent nucleotides. The purified cDNA solution was vacuum-concentrated to about 10 µl. The cDNA solution (9.4 µl) and 3.3 µl 20x saline sodium citrate (SSC) were combined and denatured at 95 °C for 5 min. The mixture was cooled at room temperature, then 0.65 µl 10 % (w/v) SDS was added.
The microarrays were hybridized, washed and scanned as described previously (Ehira et al., 2003). The relative expression ratio for each oligoDNA spot was defined as the normalized fluorescence intensity for each DNA spot at each time point relative to that at time zero (before dehydration). The experiments were performed twice independently, with different combinations of Cy dyes. Thus, the relative ratio for each DNA spot is represented by four measurements.
The means and standard deviations of the ratios of the transcript levels at each time point relative to those of the control were calculated as base-2 logarithms. A gene was judged to be upregulated when its mean relative ratio was greater than 1.6 (equivalent to threefold induction) with P value (Student's one-sample t test against 0) of less than 0.01. A gene was judged to be downregulated when its mean relative ratio was less than –1.6 (equivalent to threefold repression) with P value (Student's one-sample t test against 0) less than 0.01.
The microarray data have been deposited in the KEGG expression database (http://www.genome.jp/kegg/expression/), with the following accession numbers. Anabaena PCC 7120 (wild type strain): dehydration for 3 h, ex0001765-0001768; dehydration for 9 h, ex0001769-0001772; dehydration for 24 h, ex0001773-0001776; rehydration for 20 min, ex0001777-0001780; rehydration for 1 h, ex0001781-0001784; rehydration for 3 h, ex0001785-0001788. Anabaena ancrpB disruption mutant: dehydration for 24 h, ex0001789-0001792; rehydration for 20 min, ex0001793-0001796; rehydration for 1 h, ex0001797-0001800; rehydration for 3 h, ex0001801-0001804. Anabaena alr0618 disruption mutant: dehydration for 24 h, ex0001805-0001808; rehydration for 20 min, ex0001809-0001812; rehydration for 1 h, ex0001813-0001816; rehydration for 3 h, ex0001817-0001820.
Inactivation of ancrpB and alr0618.
To inactivate the gene ancrpB (alr2325) (Suzuki et al., 2004), a DNA fragment from the ancrpB region of Anabaena PCC 7120 was amplified by PCR using primers 5'-ACTACTCAATTTACCCTC-3' and 5'-CCCGGGCGTTCTCTAGAAATACTG-3' or 5'-CCCGGGGCATCATAGAATGCTGCA-3' and 5'-GTCAAGAATTGGTGATGA-3'. Their respective PCR products were cloned into the pGEM-T Easy vector (Promega). Both plasmids were digested with ScaI and SmaI, and were ligated together, generating a plasmid with a new SmaI restriction site at the centre of ancrpB. The 
Spr-Smr cassette excised with SmaI was inserted into the SmaI site present in the inserted DNA fragment of ancrpB. The EcoRI fragment from this plasmid was blunted with Klenow fragment and then ligated into vector pRL271 (Cai & Wolk, 1990) digested with NaeI.
To inactivate the gene alr0618, a DNA fragment from the alr0618 region of Anabaena PCC 7120 was amplified by PCR using primers 5'-AAGCTGCGAGGATTGGTCTA-3' and 5'-GGATCCCGTGAAATTTGTTCCAGGG-3' or 5'-GGATCCGCCGAGAAATTGAATGCGAT-3' and 5'-TCTGAGCATACTTCCACTCG-3'. Their respective PCR products were cloned into the pGEM-T Easy vector. Both plasmids were digested with PstI and BamHI, and were ligated together, generating a plasmid with a new BamHI restriction site at the centre of alr0618. The Spr-Smr cassette excised with BamHI was inserted into the BamHI site present in the inserted DNA fragment of alr0618. The fragment digested from this plasmid with PstI and NsiI was ligated into vector pRL271 (Cai & Wolk, 1990) digested with PstI.
ancrpB and alr0618 were inactivated with sacB-mediated positive selection for double recombination (Cai & Wolk, 1990). Transformation of Anabaena PCC 7120 was performed by a conjugation method (Elhai & Wolk, 1988), and both single and double recombinations were confirmed by PCR. Completely segregated clones of the mutants were used for the study.
Northern blotting analysis.
This was carried out as described previously (Higo et al., 2006). The cmpA probe was synthesized with a DIG PCR synthesis kit (Roche Molecular Biochemicals) using primers 5'-CCCTTCACAGCTGCTCAAAC-3' and 5'-GCCATGAAACCGACTTTGTC-3'.
Measurement of oxygen-evolving activity.
Oxygen-evolving activity was measured in intact cells with or without 5 mM NaHCO3, 10 mM HEPES (pH 7.5), using a Clark-type oxygen electrode (Rank Brothers) at a light intensity of 1000 µmol photons m–2 s–1 at 30 °C. Light was provided by a halogen lamp. The absorbance of chlorophyll extracted by methanol was measured at 665 nm and the chlorophyll concentration was calculated from the equation 1 A665 unit=13.42 µg chlorophyll ml–1 (Mackinney, 1941).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
We found that the expression of 399 genes was upregulated during dehydration and the expression of 769 genes was upregulated during rehydration when compared with their expression levels before dehydration. Among those upregulated genes, 292 genes overlapped (Fig. 1a and Supplementary Table S1, available with the online version of this paper). The genes induced under both conditions were classified into two patterns in terms of the time of maximum gene expression. (1) More than half of them showed maximum gene expression not only in 3 h or 3–24 h of dehydration but also in 1–3 h of rehydration. Many of these genes encode hypothetical and unknown proteins, while genes for trehalose metabolism were also included in this category (Higo et al., 2006). (2) The rest of the genes showed maximum expression between late dehydration and early rehydration. Among these genes, those for chaperones and proteases were markedly upregulated upon rehydration, suggesting that the control of protein quality is important after rehydration.
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and Supplementary Table S2). Most of these genes are involved in various cellular processes, such as photosynthesis, ATP synthesis, nitrogen fixation, cell wall synthesis and translation, which are essential for growth. The transcription levels of the downregulated genes were restored gradually during the rehydration period (1–3 h). Genes for ATP synthesis, photosystem II and ribosomal proteins recovered early, whereas genes for photosystem I and nitrogen fixation recovered later.
Dehydration-specific gene expression change
To exclude the genes with obscure peaks of up- or downregulation, the mean ratios at dehydration were compared with those at rehydration. We assessed whether they differed significantly using Student's two-sample t test. Of the 107 genes that were judged to be upregulated during dehydration (Fig. 1a), only 29 clearly showed maximum transcriptional level during dehydration (Supplementary Table S3). Many of these showed maximum transcriptional level during later dehydration (9–24 h). Of these, 26 genes encode hypothetical or unknown proteins. The rest encode thioredoxin, alcohol dehydrogenase and transglycosylase. In contrast, of the 299 genes downregulated during dehydration (Fig. 1b), 133 clearly showed minimum transcriptional levels during dehydration (Supplementary Table S4). Gene expression of many of those that had been downregulated after 3 h of dehydration were restored immediately after rehydration. Representatives of these include genes encoding ribulose bisphosphate carboxylase (RuBisCO), FtsZ and some ribosomal proteins. Expression of genes involved in central cellular energy metabolism was restored in a stepwise manner.
Dynamic transcriptional response to rehydration
Of the 477 genes that were upregulated during rehydration (Fig. 1a), 259 clearly showed maximum transcriptional level in the rehydration phase (Supplementary Table S5). Many of these genes showed rapid and transient upregulation with a maximum transcriptional level within 1 h of rehydration. These 259 genes were classified into the following functional groups: (a) protein folding and degradation; (b) DNA repair; (c) metabolism; (d) signal transduction; and (e) other, hypothetical and unknown function. It was noted that expression of a gene orthologue to Synechocystis sp. PCC 6803 hik34, the product of which regulates genes for chaperones and proteases (Paithoonrangsarid et al., 2004; Suzuki et al., 2005), was upregulated upon rehydration.
Forty genes upregulated on rehydration were also upregulated by nitrogen depletion (Ehira & Ohmori, 2006). Nitrogen metabolism in cyanobacteria is generally regulated by NtcA, a global nitrogen-responsive transcriptional regulator protein (Herrero et al., 2001), and ntcA was downregulated during dehydration and was also slightly downregulated during rehydration (data not shown). The gene expression patterns of many NtcA regulons and the genes involved in heterocyst formation were similar to that of ntcA. This suggests that genes upregulated during both rehydration and nitrogen depletion are independent of NtcA regulation.
As many as 57 genes related to various cellular metabolic reactions were sequentially upregulated during rehydration (Fig. 2). Quinolinate synthase (NadA) encoded by all4673, PncB encoded by alr2482, and NadD encoded by alr2483 are required for NAD biosynthesis (Gerdes et al., 2006), suggesting the depletion of the NAD pool during rehydration. Other genes markedly upregulated during rehydration were the suf operon (alr2492–alr2495), which has been shown to function in the assembly of iron–sulfur clusters under conditions of oxidative stress (Nachin et al., 2001). Rewetting the cells would cause oxidative stress (Potts, 1994). The remaining genes are involved in respiration (cytochrome c oxidase), the pentose phosphate pathway (ribulose-phosphate 3-epimerase), lipid synthesis (diacylglycerol kinase), and nitrogen and amino acid metabolism. Of the 203 genes that were downregulated during rehydration (Fig. 1b), only 13 showed minimum transcriptional level in the rehydration phase (Supplementary Table S6).
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) and six belong to a transcriptional repressor family. The remaining two genes, ancrpB and alr0618, encode CRP family transcriptional activators, and the former gene product binds to cAMP (Suzuki et al., 2004).
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). An HMMSEARCH with the HMMER2.2 software package (Eddy, 1998) against the C-terminal sequence of AnCrpB using the CRP Pfam profile (PF00325) resulted in a negative bit score, but in an E-value of 10–3. This means that the C-terminal sequence of AnCrpB is a distant homologue of the DNA-binding CRP family.
As regards the N-terminal sequence of both AnCrpB and Alr0618, they both possess a cNMP-binding domain. AnCrpB has a cAMP-binding motif and it binds cAMP (Suzuki et al., 2004). In contrast, Alr0618 does not have the motif, suggesting that this protein does not bind cAMP.
Effect of ancrpB and alr0618 disruption on gene expression during rehydration
To identify the genes regulated by AnCrpB and Alr0618, DNA microarray analysis was performed using disruptants of the corresponding genes. The genes that were upregulated by rehydration in the wild-type strain, but not in the ancrpB disruptant, are shown in Table 2. Twenty-two of 33 genes were upregulated after 20 min of rehydration only in the wild-type strain (Table 2), suggesting that ancrpB is involved in gene expression at early rehydration. The expression of five genes was upregulated by nitrogen deprivation (Table 2) and that of another five genes was also slightly upregulated by nitrogen deprivation (Ehira & Ohmori, 2006). This suggests that AnCrpB regulates the expression of genes related to nitrogen depletion.
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shows that the expression of 20 genes was upregulated by rehydration in the wild-type strain, but not in the alr0618 disruptant. The expression patterns of alr4157 (ndhD3)–alr4158 (cupA), and alr2877 (cmpA)–alr2878 (cmpB), encoding components of the NdhD3/NdhF3/CupA NAD(P)H dehydrogenase complex essential for growth under low-CO2 conditions and components of the high-affinity bicarbonate transporter, respectively, were similar to that of alr4156 (ndhF3), although the standard deviations were rather high (Supplementary Table S7). Northern blotting analysis using a cmpA probe confirmed that the entire cmp operon (alr28–77-alr2880) is upregulated by rehydration in the wild-type strain, but not in the alr0618 disruptant (Fig. 3). These genes in two gene clusters are orthlogues of genes induced under inorganic carbon limitation in Synechocystis sp. PCC 6803 (Wang et al., 2004). It is suggested that Alr0618 regulates the expression of genes related to inorganic carbon uptake.
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). In the wild-type strain, approximately 30 % of the oxygen-evolving activity was restored within 30 min of rehydration. This activity gradually returned to the level before rehydration within 24 h (data not shown).
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). Before dehydration, this activity was the same amongst all these strains. In the absence of bicarbonate, during the first 2 h, oxygen-evolving activity of the ancrpB disruptant was only slightly lower than the wild-type strain, whilst that of the alr0618 disruptant was similar to the wild-type (Fig. 4a). However, the oxygen-evolving activities in the ancrpB and the alr0618 disruptants were 60–75 % of that in the wild-type strain after 2 h of rehydration. In the presence of bicarbonate, there was no difference in oxygen-evolving activity between the wild-type and the alr0618 disruptant, while the activity of the ancrpB disruptant was lower than in the wild-type strain (Fig. 4b). These results suggest that these CRP family transcription factors are required for the recovery of photosynthetic activity during late rehydration. alr0618 is related to inorganic carbon uptake in particular. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These genes are thought to be general stress-responsive genes, involved in switching from a metabolism directed toward maximal growth to a maintenance metabolism (Hengge-Aronis, 2002). In dry yeast, ‘stationary-phase-essential genes’ and those induced during both desiccation and rehydration are similar (Singh et al., 2005). These genes might not be stimulated by a specific environmental stress, but rather by reduced metabolic states (Singh et al., 2005).
The results of the present work show that the downregulation of genes is marked during dehydration, whereas the upregulation of genes is marked during rehydration. This suggests that endurance of a stress is important during dehydration, whereas the rapid repair of damage and the resumption of metabolism are important during rehydration. A striking feature of the upregulation of gene expression during rehydration is the dynamic changes, a response that is markedly different from the upregulation of genes during dehydration. A feature of the latter was a gradual change and persistence (Katoh et al., 2004). These results reflect a difference in the dehydration and rehydration processes, the former being gradual, and the latter rapid.
In this study, the Anabaena cells were dehydrated for 24 h and then rehydrated. Previous data showed that the wet weight of the cells was constant after 9 h of dehydration (Katoh et al., 2004) and the viability of the wild-type cells was 64 % at 24 h (Higo et al., 2006). In the wild-type strain, approximately 30 % of the oxygen-evolving activity recovered within 30 min of rehydration, then there was further gradual recovery during rehydration. The activity returned to the level before rehydration within 24 h (data not shown). Tamaru et al. (2005) reported that while N. commune colonies with EPS recovered 100 % of oxygen-evolving activity rapidly after rehydration following overnight air-drying, EPS-depleted cells recovered only 30 % of the activity, a level similar to that in Anabaena PCC 7120, which contains little EPS. These results suggest that desiccation tolerance attributed to intracellular mechanisms is similar between Anabaena PCC 7120 and N. commune.
Repression of cellular metabolism during dehydration and resumption of it following rehydration
The repression of cellular metabolism during desiccation is an essential mechanism for desiccation tolerance based on the avoidance of reactive oxygen species (ROS) formation, and is not just an effect of water removal (Oliver et al., 2001). Indeed, expression of genes involved in central cellular energy metabolism was downregulated during dehydration in Anabaena PCC 7120. First, genes encoding components of photosystem II, ATP synthesis and RuBisCo were downregulated. Then, expression of genes encoding components of photosystem I and nitrogen fixation were downregulated before drying of cells was complete (Fig. 5). It would thus be reasonable to suggest that transcription of genes involved in electron-transfer processes such as photosystem II, which is a main source of ROS, were downregulated first. Downregulation of genes involved in nitrogen fixation would follow after a decrease in ATP and reducing power.
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). Respiratory activity recovered consistently within 1 h of rehydration (data not shown), whereas the recovery of net photosynthetic activity required more than 5 h of rehydration (Fig. 4). It has been reported that after rehydration, cells of N. commune resume respiration, photosynthesis and nitrogen fixation in a stepwise, coordinated manner (Scherer et al., 1984), as well as ATP synthesis (Scherer et al., 1986) and lipid biosynthesis (Taranto et al., 1993). Anabaena PCC 7120 cells should also recover their cellular activities in a stepwise, coordinated manner after rehydration, like N. commune. ATP supplied by the respiratory chain would be necessary for the recovery of fundamental cellular mechanisms including photosynthesis immediately after rehydration. Then photosystem I would supply ATP and reducing power for the resumption of nitrogen fixation. With regard to lipid biosynthesis, the expression of all0270, encoding diacylglycerol kinase (DAGK) is upregulated upon rehydration (Supplementary Table S5). DAGK catalyses the formation of phosphatidic acid from diacylglycerol and ATP, an important step in phospholipid biosynthesis. DAGK would be involved in the rapid biosynthesis of lipids induced upon rehydration, as in N. commune (Taranto et al., 1993).
The expression of many genes involved in cellular metabolism is also upregulated in response to rehydration (Supplementary Table S5). Among these, the expression of the genes of the NAD biosynthetic pathway, alr2482, alr2483 and all4673, was markedly upregulated upon rehydration (Fig. 2). NAD is used by DNA ligase, which is required for DNA repair (Eisen & Hanawalt, 1999). The expression of genes for DNA repair was upregulated after rehydration (Supplementary Table S5), suggesting that NAD is consumed to repair damaged DNA, and must be supplied via the NAD biosynthetic pathway.
Role of the two CRP family transcriptional regulators during rehydration
It has been suggested that AnCrpB regulates the expression of some genes induced by nitrogen depletion (Table 2). Heterocysts are more sensitive to desiccation than are vegetative cells (Potts & Bowman, 1985) and the recovery of nitrogenase activity during rehydration is slow in N. commune (Scherer et al., 1984). Accordingly, a shortage of nitrogen supply should result in nitrogen depletion in Anabaena cells at early rehydration. Considering that the expression of ntcA, a global nitrogen-responsive transcriptional regulator gene, is downregulated during rehydration and that there was no NtcA consensus sequence (Herrero et al., 2001) upstream of the genes listed in Table 2, AnCrpB may perform a regulatory role under nitrogen depletion, independent of NtcA. Otherwise, AnCrpB may be involved in a stress response common to both nitrogen depletion and rehydration.
The expression of alr4156, a gene induced by CO2 limitation (Wang et al., 2004), is upregulated after rehydration in the wild-type strain, while it is not upregulated in the alr0618 disruptant. The expression patterns of alr4157 and alr4158, which are also induced by CO2 limitation, were similar to that of alr4156 (Supplementary Table S7). Northern blotting clarified that expression of the cmp operon is upregulated after rehydration in the wild-type strain while it is not upregulated in the alr0618 disruptant (Fig. 3). These genes in two gene clusters encode a component of NAD(P)H dehydrogenase essential for low-CO2 growth and high-affinity bicarbonate uptake, respectively (Shibata et al., 2001). In addition, oxygen-evolving activity during rehydration in the alr0618 disruptant was lower than that in the wild-type strain only when bicarbonate was not added (Fig. 4). These results suggest that alr0618 is involved in inorganic carbon uptake.
The upregulation of alr0965, all4590, alr4057 and alr5223 expression during rehydration was abolished in the alr0618 disruptant, as well as in the ancrpB disruptant. Upregulation of nirA, encoding nitrate reductase, and nrtA and nrtD, encoding components of the nitrate transporter, was also abolished in the alr0618 disruptant. These results suggest the co-regulation of genes involved in nitrogen and carbon metabolism.
In conclusion, the gene expression response during rehydration is dynamic, whereas that during dehydration is not. Fig. 5 shows a schematic model of the transcriptional changes in the expression of metabolic genes during dehydration and rehydration. During rehydration, the expression of genes downregulated during dehydration is restored in Anabaena PCC 7120 cells. At the same time, the expression of various metabolic genes involved in NAD depletion, nitrogen depletion and CO2 limitation, as well as genes for the repair of DNA and proteins, is upregulated. It is suggested that two CRP family transcriptional regulators, AnCrpB and Alr0618, are required for the coordinated resumption of nitrogen and inorganic carbon metabolism during rehydration.
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ACKNOWLEDGEMENTS |
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Edited by: D. J. Scanlan
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Received 23 April 2007;
revised 12 June 2007;
accepted 30 July 2007.
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, Anabaena PCC 7120; 
, ancrpB disruptant; 
, alr0618 disruptant. The results are means±SD (n=3).