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 cyanobacteriumSynechococcus 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 toS. 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 whichpurF is an example, have expression peaks approximately 12 h out of phase with class 1 genes. A screen of Tn5 mutants for those in whichpurF phasing is altered revealed a mutant that carries an insertion in theopcA 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 thepurF promoter is not altered byopcA 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 inS. elongatus and that it has a role outside of its function in activating glucose-6-phosphate dehydrogenase. TheopcA gene, expected to be cotranscribed withfbp andzwf , was shown to have its own class 2 promoter, whereas thefbp 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.