Biochemical evidence for a timing mechanism in prochlorococcus

Abstract

Organisms coordinate biological activities into daily cycles using an internal circadian clock. The circadian oscillator proteins KaiA, KaiB, and KaiC are widely believed to underlie 24-h oscillations of gene expression in cyanobacteria. However, a group of very abundant cyanobacteria, namely, marine Prochlorococcus species, lost the third oscillator component, KaiA, during evolution. We demonstrate here that the remaining Kai proteins fulfill their known biochemical functions, although KaiC is hyperphosphorylated by default in this system. These data provide biochemical support for the observed evolutionary reduction of the clock locus in Prochlorococcus and are consistent with a model in which a mechanism that is less robust than the well-characterized KaiABC protein clock of Synechococcus is sufficient for biological timing in the very stable environment that Prochlorococcus inhabits.

FIG. 1.

(A) Sequence alignment of KaiC C-terminal regions from Prochlorococcus sp. strain MED4 (ProMED4), Prochlorococcus sp. strain MIT9313 (ProMIT9313), Synechococcus elongatus PCC 7942 (SynPCC7942), and “Thermosynechococcus elongatus” BP-1 (Thermosyn), where S431 and T432 (indicated by “x”), the sites of phosphorylation in S. elongatus, are conserved in Prochlorococcus sp. strain MED4 as S427 and T428, respectively. Several residues differ within the A-loop (indicated by “o”) and the C-tail between Prochlorococcus and Synechococcus strains. (B) NanoLC-MS/MS analysis of ProKaiC (purified in the presence of 0.2 mM ATP) revealed the presence of the doubly phosphorylated form pS427/pT428 in the top band and of the singly phosphorylated form in the middle band, respectively. No phosphopeptides could be detected in the faster-migrating band. (C) Dephosphorylation of ProKaiC with λ protein phosphatase (+) of two different preparations (in the presence of 0.2 mM ATP [left] and 0.5 mM ATP [right] during protein purification). After staining, three bands of different mobilities were detected using a high-resolution gel system.

FIG. 2.

(A) Pronounced autokinase activity of ProKaiC in comparison to SynKaiC. ProKaiC and SynKaiC were incubated with [γ-³²]ATP either with (+) or without (−) SynKaiA. (B) No effect of KaiB on the dephosphorylation of ProKaiC was detected, but an effect on SynKaiC dephosphorylation was detected.

FIG. 3.

ProKaiB reduces the ATPase activity of ProKaiC. ATPase assays were performed using the malachite green colorimetric method. Aliquots were sampled every 2 h. Assay buffer served as a negative control.

FIG. 4.

Scheme illustrating a putative molecular mechanism for the circadian clock in Prochlorococcus sp. strain MED4 in comparison to S. elongatus. The scheme is adapted from data described previously by Dong and Golden (2), with permission of the publisher. The homologous components that are present or absent (red crosses) in Prochlorococcus sp. strain MED4 are indicated. In the input pathway, CikA and Pex are missing in Prochlorococcus sp. strain MED4, whereas a gene, PMM1560, homologous to LdpA was found by a BLASTp search against the genome. The central timing mechanism of Prochlorococcus sp. strain MED4 lacks the KaiA component. In the output pathway, SasA (PMM1077) and RpaA (PMM0128) might influence DNA topology by underlying global gene expression rhythms. An alternative pathway via LabA was recently suggested for S. elongatus (25) but is missing in Prochlorococcus sp. strain MED4. Solid lines indicate direct effects, and dotted lines indicate indirect or unknown mechanisms. Arrows indicate the direction of signaling. Blunt ends represent inhibition, and circle ends indicate an unspecific direction.