ldpA encodes an iron-sulfur protein involved in light-dependent modulation of the circadian period in the cyanobacterium Synechococcus elongatus PCC 7942

Abstract

We generated random transposon insertion mutants to identify genes involved in light input pathways to the circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942. Two mutants, AMC408-M1 and AMC408-M2, were isolated that responded to a 5-h dark pulse differently from the wild-type strain. The two mutants carried independent transposon insertions in an open reading frame here named ldpA (for light-dependent period). Although the mutants were isolated by a phase shift screening protocol, the actual defect is a conditional alteration in the circadian period. The mutants retain the wild-type ability to phase shift the circadian gene expression (bioluminescent reporter) rhythm if the timing of administration of the dark pulse is corrected for a 1-h shortening of the circadian period in the mutant. Further analysis indicated that the conditional short-period mutant phenotype results from insensitivity to light gradients that normally modulate the circadian period in S. elongatus, lengthening the period at low light intensities. The ldpA gene encodes a polypeptide that predicts a 7Fe-8S cluster-binding motif expected to be involved in redox reactions. We suggest that the LdpA protein modulates the circadian clock as an indirect function of light intensity by sensing changes in cellular physiology.

FIG. 1.

Traces of circadian rhythms of bioluminescence from PpurF::luxAB reporter strains. Symbols: diamonds, wild type; triangles, mutant AMC408-M1; squares, mutant AMC408-M2. Hour zero in LL indicates the time point of the end of the 12-h dark interval used for initial synchronization. The black bar indicates the time of application of a 5-h dark pulse for phase resetting.

FIG. 2.

Physical map of the ldpA region of the S. elongatus chromosome and plasmids used to reconstruct intact and disrupted ldpA alleles. Restriction sites are indicated by the following abbreviations: E, EcoRI; Pv, PvuII; Ps, PstI; An, AlwNI; Bg, BglII; Hp, HpaI. Insertion sites of the transposon and Gm^r cassette are indicated by filled and open triangles, respectively. Broken lines indicate regions derived from the transposon. Plasmids pAM2180 and pAM2181 were recovered by restriction digestion and circularization from genomic DNA of mutants AMC408-M1 and AMC408-M2, respectively.

FIG. 3.

Comparison of the deduced amino acid sequence encoded by the ldpA gene of S. elongatus PCC 7942 and ferredoxins with known 7Fe-8S centers. LdpA, GenBank accession no. AY136759; T. thermophilus ferredoxin, SwissProt accession no. P03942; Bacillus schlegelii ferredoxin, SwissProt accession no. Q45560; A. vinelandii FdI, SwissProt accession no. P00214. Only the Fe-S center domain is shown. Conserved Cys residues are highlighted, and corresponding Fe-S centers are indicated by brackets. The PDB codes for the ferredoxins from A. vinelandii, T. thermophilus, and B. schlegelii are 1F5B, 1H98, and 1BC6, respectively. Pairwise sequence similarities between LdpA and other ferredoxins were assessed by an alignment-independent PRSS test. Statistical significance (P values) for the pairwise sequence comparison is indicated on the right of the sequence alignment.

FIG. 4.

PRCs for 5-h dark pulses in the wild-type strain (solid circles) and ldpA mutants AMC694 (○) and AMC695 (▵). PRCs for duplicate samples of each strain are shown. Circadian time (CT) is a normalization for the difference between the circadian periods of the strains; 1 circadian h = 1/24 of a full circadian cycle. Positive values represent phase advances, and negative values represent phase delays.

FIG. 5.

Effect of white light fluence on the FRP. Panels A and B show the FRPs of the wild type (A) and C22a (B) (•) and those genetic backgrounds with ldpA disrupted (○). Panels C and D show the FRPs of C22a (C) and C22a with ldpA disrupted (D) with (▵, ▴) and without DCMU addition (○, •). DCMU was added to media at a final concentration of 0.2 μM at the beginning of a 12-h dark interval. Error bars indicate standard deviations (n = 8).

FIG. 6.

Resetting of circadian rhythms by DCMU. Cyanobacterial suspensions (3 ml) with an OD₇₃₀ of 0.3 were exposed a synchronizing 12-h dark interval, and then 1 μM DCMU (A) or 0.05% ethanol (solvent used for DCMU; B) was added. After incubation for 12 h under standard light conditions, cells were collected by centrifugation, washed twice with fresh BG-11 M, and suspended in 3 ml of BG-11 M. Samples (10 μl) were inoculated onto sample plates, and circadian rhythms were measured. The interval of DCMU treatment is indicated by the gray bar in panel B.