BACTERIAL LUCIFERASE AS A REPORTER OF CIRCADIAN GENE-EXPRESSION IN CYANOBACTERIA

To allow continuous monitoring of the circadian clock in cyanobacteria , we previously created a reporter strain (AMC149) of Synechococcus sp . strain PCC 7942 in which the promoter of the psbAI gene was fused to Vibrio harveyi luciferase structural genes (luxAB) and integrated int o the chromosome. Northern (RNA) hybridization and immunoblot analyses were performed to examine changes in abundance of the luxAB mRNA, the native psbAI mRNA, and the luciferase protein to determine whether bi oluminescence is an accurate reporter of psbAI promoter activity in AM C149. Under constant light conditions, the mRNA abundances of both lux AB and psbAI oscillated with a period of approximately 24 h for at lea st 2 days. The expression of these two genes followed the same pattern : both mRNAs peaked in the subjective morning, and their troughs occur red near the end of the subjective night. The amount of luciferase pro tein also oscillated with a period of approximately 24 h, and the prot ein rhythm is in phase with the bioluminescence rhythm. The rhythm of the luciferase mRNA phase-leads the rhythms of luciferase protein and in vivo bioluminescence by several hours. Comparable results were obta ined with a short-period mutant of AMC149. Together, these results ind icate that the bioluminescence rhythm in AMC149 is due primarily to ci rcadian oscillation of psbAI promoter activity in this cyanobacterium.