cpmA, a gene involved in an output pathway of the cyanobacterial circadian system

Abstract

We generated random mutations in Synechococcus sp. strain PCC 7942 to look for genes of output pathways in the cyanobacterial circadian system. A derivative of transposon Tn5 was introduced into the chromosomes of reporter strains in which cyanobacterial promoters drive the Vibrio harveyi luxAB genes and produce an oscillation of bioluminescence as a function of circadian gene expression. Among low-amplitude mutants, one mutant, tnp6, had an insertion in a 780-bp open reading frame. The tnp6 mutation produced an altered circadian phasing phenotype in the expression rhythms of psbAI::luxAB, psbAII::luxAB, and kaiA::luxAB but had no or little effect on those of psbAIII::luxAB, purF::luxAB, kaiB::luxAB, rpoD2::luxAB, ndhD::luxAB, and conII::luxAB. This suggests that the interrupted gene in tnp6, named cpmA (circadian phase modifier), is part of a circadian output pathway that regulates the expression rhythms of psbAI, psbAII, and kaiA.

FIG. 1

Bioluminescence traces from the tnp6 mutant of Synechococcus AMC149. Bioluminescence was measured from streaks on agar plates of wild-type AMC149 (A); original tnp6 mutant (B); and recreated tnp6 mutant, tnp6K (C). Time shown on the x axis refers to hours in LL after a synchronizing 12-h dark incubation. The y axis indicates bioluminescence (counts/3-min bin) from baseline of 0 to maxima of 101,000 (panel A), 57,300 (panel B), and 64,100 (panel C) as detected by a cooled charge-coupled device camera (20).

FIG. 2

Chromosomal maps of specific loci from recombinant cyanobacterial strains. Shown are physical maps of original tnp6 mutant of Synechococcus (A), the inactivated cpmA locus in which an NruI fragment was replaced by a Km^r gene cartridge (B), and the NSII loci in AMC540 and AMC540(cpmA::Km^r) (C).

FIG. 3

Comparison of amino acid sequences including that deduced from the cpmA gene of Synechococcus sp. strain PCC 7942 (Synco-CpmA), putative cpmA homologs from Synechocystis sp. strain PCC 6803 (Syncy-CpmA), M. jannaschii (Metco-CpmA), M. thermoautotrophicum (Metba-CpmA), and A. fulgidus (Arcog-CpmA), and the sequences of PurE proteins of Synechocystis sp. strain PCC 6803 (Syncy-PurE), M. jannaschii (Metco-PurE), Mycobacterium leprae (Mycba-PurE), and E. coli (Esche-PurE). Residues identical among all CpmA (but not all PurE) sequences are boxed; those common to all aligned sequences are shown in boldface.

FIG. 4

Loss of cpmA function causes low amplitude and early phase angle in the psbAI::luxAB bioluminescence rhythm. Bioluminescence traces were obtained from AMC412, the wild-type strain (A), AMC412(cpmA::Km^r), the mutant in which the cpmA gene was inactivated by the replacement of an internal NruI fragment with a Km^r gene cartridge (B), and AMC540(cpmA::Km), the cpmA mutant that carries an ectopic wild-type allele of cpmA in trans (C).

FIG. 5

Effect of cpmA inactivation on the expression rhythms of various reporter genes. Closed circles, wild-type traces; open circles, cpmA mutant traces. The x axis is labeled as for Fig. 1. The y axis values are counts per second. A representative trace is shown for each genotype, and the identity of the Synechococcus gene fused to luxAB is indicated for each panel. The fraction of independent experiments and the fraction of total independent colonies for which marked phase-change phenotypes (several hours’ phase angle difference) were observed, respectively, follow in parentheses for each reporter strain: psbAI::luxAB (9/10; 37/48), psbAII::luxAB (6/10; 22/46), psbAIII::luxAB (1/6; 4/31), purF::luxAB (0/9; 0/41), kaiA::luxAB (8/9; 38/42), kaiB::luxAB (0/10; 0/47), rpoD2::luxAB (3/9; 12/31), ndhD::luxAB (1/9; 1/37), and conII::luxAB (2/7; 10/35) reporter fusions.