Gene Expression in the Cyanobacterium Anabaena sp. PCC7120 under Desiccation H. Katoh1,2, R.K. Asthana3 and M. Ohmori1 (1) Department of Life Sciences (Biology), University of Tokyo, Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan (2) Bio-oriented Technology Research Advancement Institution (BRAIN), 1-40-2 Nisshin, Saitama, Saitama 331-8537, Japan (3) Department of Botany, Banaras Hindu University, B.H.U. Varanasi-221 005, India Received: 26 February 2003 / Accepted: 12 August 2003 / Online publication: 2 February 2004
Abstract
The N2-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K+ transporting system are upregulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.
Introduction
Microarray technology has emerged as a crucial device for the analysis of global-scale gene expression [7, 33]. Determination of gene expressions under extreme envi-
ronments, including space flight, offers useful information about the acclimation of organisms to new environments. The profiling of plant-gene expressions has been based on a cDNA microarray technique [35]. Recently, the genome sequence of the filamentous, heterocystous cyanobacterium Anabaena (Nostoc) sp. PCC7120 has been determined (6.4 Mbp plus 798 kbp of six plasmids) [14], and genome-wide computer analyses have already been carried out [25]. We have made a DNA microarray for Anabaena using the sequencing clones (3 to 4 kbp). Cyanobacteria, the photosynthetic, O2-evolving proarchconservate, grow in diverse habitats ranging from tropical to polar regions. They spread all over the earth in ancient times and have made our planet oxidative. The extent of their desiccation tolerance constituted a fundamental component of their survival strategy owing to their archaic origin. However, acclimation studies in cyanobacteria using DNA microarray technique have been mainly confined to salt and hyperosmotic stress [8, 15, 38], high light [11], and the transition from a light to a dark regime [10]. Although the possible mechanisms of desiccation tolerance have been reported in the cyanobacterium Nostoc commune [28], genome-wide gene expression related to the process is still awaiting investigation. We have recently isolated a terrestrial, desiccation-tolerant cyanobacterium, Nostoc sp. HK-01, whose 16S rRNA sequence was very similar to that of Anabaena sp. PCC7120 [16]. In the present experiment, we determined the gene expression in desiccated Anabaena sp. PCC7120 using a DNA microarray technique. Methods and Culture Conditions. Nitrogen-fixing Anabaena sp. PCC7120 was grown at 31C in BG11 medium [36] with 20 mM HEPES–NaOH (pH 7.5) at 40
Organism Correspondence to: M. Ohmori; E-mail:
[email protected]. ac.jp
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DOI: 10.1007/s00248-003-1043-6
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lE m)2 s)1 under continuous fluorescent illumination. Cells were grown in volumes of 50 mL in culture tubes (3 cm dia) and bubbled with air containing CO2 (1.0%, v/ v). Cell density was estimated as A750 by a spectrophotometer (model UV-160A; Shimadzu, Kyoto, Japan). The chlorophyll concentration extracted by methanol was measured at 665 nm and then calculated with A665 1 = 13.42 lg Chl/ml [19]. Viability Check of Desiccated Anabaena. Cells were harvested by centrifugation at a cell density of A750 = 1.2 to 1.8, and the pellet was resuspended in BG11 medium at a concentration of about 80 lg Chl mL)1. The cells were then spotted on ‘‘mixed cellulose ester’’ filter paper (0.45 lm pore size, Advantec, Tokyo, Japan). The filter paper and cells were desiccated in a petri dish (90 · 15 mm) under room-light conditions (30 to 40 lE m)2 s)1) at 30C and 30% relative humidity for 10 min, 30 min, 1 h, 3 h, 6 h, 10 h, and 22 h. The filter paper and cells were then put on a BG11 agar plate, supplemented with 0.6% sodium thiosulfate, and kept illuminated (30 to 40 lE m)2 s)1) at 30C for 4 days. Viability was calculated as the numbers of colonies formed. Isolation of Total RNA. A 30-mL portion of cell culture was harvested by filtration on the filter paper at a cell density of A750 = 1.2 to 1.8 and desiccated. After the designated time, the cells were harvested, and total RNA was isolated immediately by the hot-phenol method of Mohamed and Jansson [21]. Crude total RNA was treated with 5 lU/lL DNase I (Takara, Shiga, Japan) in 100 mM sodium acetate (pH 5.0) and 5 mM MgSO4 at 37C for 1 h as described in the manual. After 50:49:1 phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation, total RNA was suspended in RNasefree ultrapure water. Preparation of Labeled cDNA. Fluorescently labeled cDNA probes were prepared from the total RNA pool by direct incorporation of fluorescent nucleotide analogues (Cy3-dUTP or Cy5-dUTP; Amersham Pharmacia, Uppsala, Sweden) during the first-strand reverse transcriptase reaction with AMV Reverse Transcriptase XL (Takara, [11]). Free fluorescent nucleotides were removed using a QIA-quick PCR purification Kit (QIAGEN, Tokyo, Japan). After phenol treatment and ethanol precipitation, the pellets were stored in the dark at )20C. Features of DNA Microarrays. Anabaena genome sequence clones ranging from 3 to 4 kbp were the kind gifts of the Kazusa DNA Research Institute [14]. Anabaena DNA fragments in the sequence clones were amplified by PCR using universal primers (5¢-GGGTTTTCCCAGTCACGAC-3¢ and 5¢-TTATGCTTCCGGCTCGTATGTTGTG-3¢), and the products (3 to 4 kbp)
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were spotted on a poly-L-lysine-coated slide glass (SPBIO-E, Hitachi Software Engineering Co., Ltd., Yokohama, Japan). The DNA microarray was irradiated by UV (2500 lJ cm)2 for 1 s), treated with 140 mM succinic anhydride, 0.1 M boric acid (pH 8.0) and 90%(v/v) 1-methyl-2-pyrrolidinone for 20 min followed by repeated washings in water (MilliQ) and soaking in boiling water for 3 min, and then in ice cooled ethanol for 3 min before drying. The DNA microarray contained 2407 spots corresponding to approximately 86% of genome DNA, and each spot was duplicated. The DNA microarray was stored in a desiccator in the dark before using. Hybridization and Washing Conditions of the DNA Microarray. For hybridization, Cy3- and Cy5-labeled
cDNA was dissolved in total 38 lL of 5· SSC (1· SSC containing 0.15 M NaCl and 0.015 M sodium citrate). After incubation at 95C for 5 min the sample was incubated at 25C for 30 min, and then 2 lL of 10%(w/v) SDS was added. The solution was spread onto the microarray surface, covered with a cover slip (Spaced Cover Glass L; Takara), and sealed with a Hybridization Cassette (Arrayit, Sunnyvale, CA, USA). Hybridization was performed overnight at 65C. Washing was performed as follows: (i) three washes in 2· SSC and 0.2% SDS at room temperature for 10 min; (ii) three washes in 0.2· SSC and 0.2% SDS at room temperature for 10 min; (iii) three washes in 0.2· SSC and 0.2% SDS at 60C for 10 min; and (iv) two washes in 0.2· SSC at room temperature for 5 min. The microarray was then dried using a spin dryer (Spin Dryer mini 2350, Wakenyaku, Co., Ltd., Kyoto, Japan). Image Acquisition and Analysis. Microarrays were scanned with two wavelengths for Cy3 (570 nm) and Cy5 (660 nm) using a laser fluorescent scanner (Scanarray 4000, GSI Lumonics, Billerica, MA, USA) with three different photomultiplier gains. Data analysis was performed using Quantarray software (GSI Lumonics). The raw data obtained with the optimum photomultiplier gain were routinely used for quantification. The fluorescence intensity of each spot in both the Cy3 and Cy5 images was quantified, and fluorescence levels of the local background were subtracted. Normalization was performed by adjusting the total signal intensity of the Cy3 and Cy5 images. Changes in the level of the transcript of each gene relative to the total level of mRNA were then calculated. The expression level of each gene under desiccation stress was indicated by desiccation/control ratio. Eight observations were recorded for each time point. All expression rates described here are shown as averages with standard deviations of eight observations. RT-PCR. To identify the genes related to desiccation, RT-PCR was performed by an RNA PCR kit
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Table 1. Primers used for RT-PCR
Gene name all0166 all0167 all0168 all0457 all0458 all0459 alr0803 alr0804 alr0805 all1058 all1059
Forward primer
Reverse primer
TCGTTCCTGGTGGTCGTT CCAATTCTATCGCCTCGC GTATTGCTGTAGTGCTGG GAAACTCGGATTGCAGGT CCACAGTTCCGCAGTCTA TTACGCCCAGTCCGCTTT GGTGGTCTATTGGGTGGA TGTTTGGTGCTGCGGCTT ACCGAACAAAAGCAACCG GGAGTAGCCGAGTTGAGT CCGTCGTCTCAAAGTCAC
GTTCAGACAGACAGGTGC CATCGAACTCCAGCAGCA GCATACCGCCAATCATAG AGGACAAGGACGAGCCAA AGCGCAACATCCAAGCAA CCGGTGACTTTTTCCAGG CCTAAACCTCCGCCTGTA ACTGGTCTTGGTTGTTGA GGACTTGATGAGATAACG AATATCCGTCCTGGGTGC GGCTTCTTCGGGATTGGT
(Takara). The primers for RT-PCR are shown in Table 1. PCR amplifications were performed with a Gene Amp PCR System 9700 (Applied Biosystems, USA) for 20 cycles each consisting of 30 s at 94C, 30 s at 54C, and 1 min at 72C. Results Viability of Anabaena Following Desiccation. We determined the relation between cell viability and the length of desiccation. Although once exposed to desiccation for up to 3 h, Anabaena cells grew at almost the same rate as when rewetted, the cells desiccated for 6 h or more showed inhibited growth. The viabilities of 6-h and 10-h desiccated cells were
