Improvement of the bioluminescence reporter system for real-time monitoring of circadian rhythms in the cyanobacterium Synechocystis sp strain PCC 6803

Circadian rhythm is a self-sustaining oscillation whose period length coincides with the 24-hour day-night cycle. A powerful tool for circadian clock research is the real-time automated bioluminescence monitoring system in which a promoter region of a clock-controlled gene is fused to a luciferase reporter gene and rhythmic regulation of the promoter activity is monitored as bioluminescence. In the present study, we greatly improved the bioluminescence reporter system in the cyanobacterium Synechocystis sp. strain PCC 6803. We fused an 805-bp promoter region of the dnaK gene seamlessly to the luy-A coding sequence and integrated the P-dnaK::luxAB fusion gene into a specific intergenic region of the Synechocystis genome (targeting site 1). The resulting new reporter strain, PdnaK::IuxAB(-), showed 12 times the bioluminescence intensity of the standard reporter strain, CFC2. Furthermore, we generated strain PdnaK::IuxAB(+), in which the PdnaK::1uxAB fusion gene and the selection-marker spectinomycin resistance gene are transcribed in opposite directions. The PdnaK::IuxAB(+) strain showed 19 times the bioluminescence intensity of strain CFC2. The procedures used to increase the bioluminescence intensity are especially useful for bioluminescence monitoring of genes with low promoter activity. In addition, these reporter constructs facilitate bioluminescence monitoring of any gene because the promoter fragments they contain can easily be replaced by digestion with unique restriction enzymes. They would therefore contribute to a genome-wide analysis of gene expression in Synechocystis.