Salt Stress and Hyperosmotic Stress Regulate the Expression of Different Sets of Genes in Synechocystis sp. PCC 6803 Yu Kanesaki,* , † Iwane Suzuki,* , † Suleyman I. Allakhverdiev,* Koji Mikami,* and Norio Murata* , † ,1 *Department of Regulation Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan; and †Department of Molecular Biomechanics, School of Life Science, Graduate University for Advanced Studies, Okazaki 444-8585, Japan Received November 30, 2001 Acclimation of microorganisms to environmental stress is closely related to the expression of various genes. We report here that salt stress and hyperos- motic stress have different effects on the cytoplasmic volume and gene expression in Synechocystis sp. PCC 6803. DNA microarray analysis indicated that salt stress strongly induced the genes for some ribosomal proteins. Hyperosmotic stress strongly induced the genes for 3-ketoacyl-acyl carrier protein reductase and rare lipoprotein A. Genes whose expression was induced both by salt stress and by hyperosmotic stress included those for heat-shock proteins and the en- zymes for the synthesis of glucosylglycerol. We also found that each kind of stress induced a number of genes for proteins of unknown function. Our findings suggest that Synechocystis recognizes salt stress and hyperosmotic stress as different stimuli, although mechanisms common to the responses to each form of stress might also contribute to gene expression. © 2002 Elsevier Science Key Words: DNA microarray; cyanobacteria; salt stress; hyperosmotic stress. Microorganisms including cyanobacteria acclimate to various kinds of environmental stress by regulating the expression of numerous stress-inducible genes (1– 3). For example, when the cyanobacterium Synechocys- tis sp. PCC 6803 (hereafter Synechocystis) is exposed to salt stress, expression of the following genes is in- duced: the ggpS gene for glucosylglycerolphosphate synthase (4); the crh gene for RNA helicase (3); the isiA gene for iron-stress-inducible protein A (5); the isiB gene for flavodoxin (5) and the petH gene for ferre- doxin:NADP + reductase (6). The terms salt stress and hyperosmotic stress have often been used in a confusing manner, such that genes induced upon exposure of organisms to high concentra- tions of NaCl have sometimes been defined as osmo- stressed genes (7, 8). However, the accumulated evi- dence suggests that the two kinds of stress are perceived as different signals. Incubation of the cya- nobacterium Synechococcus sp. PCC 7942 in medium supplemented with 1 M sorbitol decreases the cytoplas- mic volume to 45% of the original value (9), whereas incubation in 0.5 M NaCl only decreases the volume to 85% of the original value (10). These findings suggest that cyanobacterial cells might respond to salt stress and hyperosmotic stress in different ways. We won- dered whether these two kinds of stress might induce the same set or different sets of genes. To examine this issue, we used a DNA microarray to investigate gene expression in Synechocystis in response to salt stress and to hyperosmotic stress. DNA microarrays allow monitoring of changes in levels of transcripts of almost all genes in specific or- ganisms (11, 12). Such arrays have been used to exam- ine gene expression in response to various kinds of stress in Saccharomyces cerevisiae (13) and in Synecho- cystis (14, 15). In the present study, we obtained clear evidence that salt stress and hyperosmotic stress regulate different sets of genes, although expression of some genes was induced in common by both kinds of stress. Further- more, we found that expression of a number of genes for proteins of unknown function was induced or re- pressed by salt stress and hyperosmotic stress. MATERIALS AND METHODS Strain and culture conditions. A glucose-tolerant strain of Syn- echocystis sp. PCC 6803 was kindly provided by Dr. J. G. K. Williams (Du Pont de Nemours & Co., Inc., Wilmington, DE). Cells were grown at 34°C in 50 ml of BG-11 medium (16) buffered with 20 mM Hepes– 1 To whom correspondence and reprint requests should be ad- dressed at National Institute for Basic Biology, Okazaki 444-8585, Japan. Fax: (+81) 564 54 4866. E-mail: murata@nibb.ac.jp. Biochemical and Biophysical Research Communications 290, 339 –348 (2002) doi:10.1006/bbrc.2001.6201, available online at http://www.idealibrary.com on 339 0006-291X/02 $35.00 © 2002 Elsevier Science All rights reserved.