Aoki S, Kondo T, Ishiura M
National Institute for Basic Biology, Aichi, Japan.
J Bacteriol. 1995 Oct;177(19):5606-11. doi: 10.1128/jb.177.19.5606-5611.1995.
The expression of the dnaK gene in the cyanobacterium Synechocystis sp. strain PCC 6803 was continuously monitored as bioluminescence by an automated monitoring system, using the bacterial luciferase genes (luxAB) of Vibrio harveyi as a reporter of promoter activity. A dnaK-reporting bioluminescent Synechocystis strain was constructed by fusing a promoterless segment of the luxAB gene set downstream of the promoter region of the Synechocystis dnaK gene and introduction of this gene fusion into a BglII site downstream of the ndhB gene in the Synechocystis chromosome. Bioluminescence from this strain was continuously monitored and oscillated with a period of about 22 h for at least 5 days in continuous light. The phase of the rhythm was reset by the timing of the 12-h dark period administered prior to the continuous light. The period of the rhythm was temperature compensated between 25 and 35 degrees C. Thus, the bioluminescence rhythm satisfied the three criteria of circadian rhythms. Furthermore, the abundance of dnaK mRNA also oscillated with a period of about 1 day for at least 2 days in continuous light conditions, indicating circadian control of dnaK gene expression in Synechocystis sp. strain PCC 6803.
利用哈维氏弧菌的细菌荧光素酶基因(luxAB)作为启动子活性的报告基因,通过自动监测系统将蓝藻聚球藻属PCC 6803菌株中dnaK基因的表达作为生物发光进行持续监测。通过将luxAB基因的无启动子片段融合到聚球藻dnaK基因启动子区域的下游,并将该基因融合体引入聚球藻染色体中ndhB基因下游的BglII位点,构建了一个dnaK报告生物发光聚球藻菌株。在连续光照下,对该菌株的生物发光进行持续监测,其振荡周期约为22小时,至少持续5天。在连续光照之前施加12小时黑暗期的时间可重置节律的相位。节律的周期在25至35摄氏度之间进行温度补偿。因此,生物发光节律满足昼夜节律的三个标准。此外,在连续光照条件下,dnaK mRNA的丰度也至少在2天内以约1天的周期振荡,表明聚球藻属PCC 6803菌株中dnaK基因表达受昼夜节律控制。