Changes in the supercoiling of genomic DNA play an important role in the regulation of gene expression. We compared the genome-wide expression of genes in cells of the cyanobacterium Synechocystis sp. PCC 6803 when they were subjected to salt, cold, and heat stress, in the presence of novobiocin, an inhibitor of DNA gyrase, and in its absence. The analysis revealed that the expression of a large number of stress-inducible genes depends on the extent of genomic DNA supercoiling. The function of the two-component regulatory systems, which are known as sensors and transducers of salt, cold, and heat stress, depends on, and might be controlled by, the degree of supercoiling of the genomic DNA. These results suggest that stress-induced changes in superhelicity of genomic DNA provide an important permissive background for successful acclimatization of cyanobacterial cells to stress conditions.