A New Circadian Class 2 Gene, opcA, Whose Product Is Important for Reductant Production at Night in Synechococcus elongatus PCC 7942

Gene expression in the cyanobacterium Synechococcus elongatus PCC 7942 is under the control of a circadian oscillator, such that peaks and troughs of expression recur with a periodicity of about 24 h in the absence of environmental cues. This can be monitored easily as light production from luciferase gene fusions to S. elongatus promoters. All promoters seem to exhibit circadian oscillation of expression, but the phasing of peak and trough times differs among different genes. The majority of genes are designated class 1, with expression peaks near dusk or subjective dusk (the time corresponding to dusk in the absence of a diurnal cycle). A minority, of which purF is an example, have expression peaks approximately 12 h out of phase with class 1 genes. A screen of Tn5 mutants for those in which purF phasing is altered revealed a mutant that carries an insertion in the opcA gene, previously identified as essential for glucose-6-phosphate dehydrogenase function. However, a different enzymatic reporter and in vitro luciferase assays revealed that the expression pattern of the purF promoter is not altered by opcA inactivation, but rather the reduced flavin mononucleotide substrate of luciferase is limiting at the time of the natural circadian peak. The results suggest that OpcA is involved in temporally separated reductant-generating pathways in S. elongatus and that it has a role outside of its function in activating glucose-6-phosphate dehydrogenase. The opcA gene, expected to be cotranscribed with fbp and zwf, was shown to have its own class 2 promoter, whereas the fbp promoter was determined to be in class 1. Thus, opcA expression is likely to be constitutive by virtue of the activity of two promoters in nearly opposite circadian phases. Cyanobacteria, like diverse eukaryotes, possess an internal timekeeping mechanism termed a circadian clock that controls oscillations of biological functions with an endogenous period of about 24 h (2, 6, 12, 14, 34). A circadian system is conventionally divided into three parts: a central oscillator that determines the circadian period, input pathways that synchronize the phasing of the oscillator with daily environmental cycles, and output pathways that confer circadian rhythmicity on the processes that are controlled by the clock. A locus of three genes, kaiA, kaiB, and kaiC, has been identified in the cyanobacterium Synechococcus elongatus PCC 7942 that is fundamentally important for circadian rhythmicity of gene expression (16). Mutations in any of these genes can alter the circadian period or cause arhythmicity of all genes that are examined. Most or all of the genes in the S. elongatus genome are transcribed with a circadian rhythm. One demonstration of this was the insertion of promoterless Vibrio harveyi luciferase genes (luxAB) randomly throughout the genome. All bioluminescent colonies, indicating fusion of the reporter genes with a promoter, showed a circadian rhythm of light production (19). These and other data suggest that transcription is globally rhythmic in the organism. However, there is specialization in the phasing (timing of peaks and troughs), amplitude, and duration of the day with which individual genes are expressed. Most S. elongatus promoters drive luxAB with a peak of bioluminescence at dusk (in a light-dark cycle) or subjective dusk (in continuous light [LL]) and a trough at dawn or subjective