The pseudo‐receiver domain of CikA regulates the cyanobacterial circadian input pathway

Abstract

SummaryCikA (circadian input kinase) is a component of the cyanobacterial circadian clock that aids in synchronizing the endogenous oscillator with the external environment. cikA mutants of the prokaryotic circadian model organism Synechococcus elongatus PCC 7942 fail to reset the phase of the circadian rhythm of gene expression after an environmental time cue, and also exhibit reduced amplitude and shortened period of circadian oscillation. CikA has histidine protein kinase (HPK) activity that is modulated in vitro by GAF and pseudo -receiver (PsR) domains. Here we show that the PsR domain negatively regulates HPK activity in vivo and also serves as an interaction module to dock CikA at a specific subcellular location. Phenotypes conferred by alleles that encode CikA variants showed that all domains except the featureless Nterminus are required for CikA function. Overexpression of all alleles that encode the PsR domain, whether or not the HPK is functional, caused a dominant arrhythmic phenotype, whereas overexpressed variants that lack PsR did not. Subcellular localization of intact CikA identified a polar focus whereas a variant without PsR showed uniform distribution in the cell, consistent with a model in which PsR mediates interaction with other input pathway components.

Quinone sensing by the circadian input kinase of the cyanobacterial circadian clock

“…1B, lane 2). We did not detect KaiC in the fraction that copurifies with CikA-His expressed at the WT level (21), probably because the yield of eluted CikA-His is an order of magnitude lower than that of KaiC-His expressed at its WT level based on silver-stained SDS͞PAGE (data not shown). However, KaiC was found in the eluted fraction when CikA-His was overexpressed (Fig.…”

“…In both samples, CikA is present in fractions that correspond to a molecular mass of just under 440 kDa (Fig. 1C); this value is larger than the active CikA dimer of 166 kDa (21), which indicates that CikA participates in a protein complex.…”

“…A striking phenotypic feature of the cikA mutant is its inability to reset the phase of the circadian rhythm after a 5-h dark pulse, whereas WT cells respond in the next cycle with a peak that is offset by up to 12 h relative to the initial daily peak time (8). The greatest difference in circadian resetting between the WT and cikA strains is detected when the dark pulse is given at ZT8 (21). To establish the role CikA plays during the dark pulse, cikA-null and WT cells were collected before, during, and after a pulse administered at ZT8, and protein extracts were subjected to immunoblot analysis.…”

“…To identify the domain of CikA responsible for CikA sensitivity to DBMIB, we examined a collection of strains that carry defective variants of CikA encoded by point or deletion mutations in cikA (21). When various concentrations of DBMIB were added to the panel of mutants, those CikA variants that lack the GAF domain (⌬GAF) or carry a substitution in the phosphorylation site of the kinase domain (H393A) were still sensitive to DBMIB, although to a lesser degree than the WT protein (Fig.…”

“…4); thus, the PsR domain is necessary and sufficient for DBMIB sensitivity of CikA. is localized to the poles of the cell (21). To see whether specific membrane-associated or soluble factors are necessary for the effect of DBMIB on CikA, we separated cyanobacterial proteins into membrane and soluble fractions; CikA was present in both (Fig.…”