Cross-talk and regulatory interactions between the essential response regulator RpaB and cyanobacterial circadian clock output

The response regulator RpaB (regulator of phycobilisome associated B), part of an essential two-component system conserved in cyanobacteria that responds to multiple environmental signals, has recently been implicated in the control of cell dimensions and of circadian rhythms of gene expression in the model cyanobacterium Synechococcus elongatus PCC 7942. However, little is known of the molecular mechanisms that underlie RpaB functions. In this study we show that the regulation of phenotypes by RpaB is intimately connected with the activity of RpaA (regulator of phycobilisome associated A), the master regulator of circadian transcription patterns. RpaB affects RpaA activity both through control of gene expression, a function requiring an intact effector domain, and via altering RpaA phosphorylation, a function mediated through the N-terminal receiver domain of RpaB. Thus, both phosphorylation cross-talk and coregulation of target genes play a role in the genetic interactions between the RpaA and RpaB pathways. In addition, RpaB∼P levels appear critical for survival under light:dark cycles, conditions in which RpaB phosphorylation is environmentally driven independent of the circadian clock. We propose that the complex regulatory interactions between the essential and environmentally sensitive NblS-RpaB system and the SasA-RpaA clock output system integrate relevant extra- and intracellular signals to the circadian clock.

Detecting KaiC Phosphorylation Rhythms of the Cyanobacterial Circadian Oscillator In Vitro and In Vivo

The central oscillator of the cyanobacterial circadian clock is unique in the biochemical simplicity of its components and the robustness of the oscillation. The oscillator is composed of three cyanobacterial proteins: KaiA, KaiB, and KaiC. If very pure preparations of these three proteins are mixed in a test tube in the right proportions and with ATP and MgCl2, the phosphorylation states of KaiC will oscillate with a circadian period, and these states can be analyzed simply by SDS-PAGE. The purity of the proteins is critical for obtaining robust oscillation. Contaminating proteases will destroy oscillation by degradation of Kai proteins, and ATPases will attenuate robustness by consumption of ATP. Here, we provide a detailed protocol to obtain pure recombinant proteins from Escherichia coli to construct a robust cyanobacterial circadian oscillator in vitro. In addition, we present a protocol that facilitates analysis of phosphorylation states of KaiC and other phosphorylated proteins from in vivo samples.

PipX, the coactivator of NtcA, is a global regulator in cyanobacteria

To modulate the expression of genes involved in nitrogen assimilation, the cyanobacterial PII-interacting protein X (PipX) interacts with the global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance. PipX can form alternate complexes with NtcA and PII, and these interactions are stimulated and inhibited, respectively, by 2-oxoglutarate, providing a mechanistic link between PII signaling and NtcA-regulated gene expression. Here, we demonstrate that PipX is involved in a much wider interaction network. The effect of pipX alleles on transcript levels was studied by RNA sequencing of S. elongatus strains grown in the presence of either nitrate or ammonium, followed by multivariate analyses of relevant mutant/control comparisons. As a result of this process, 222 genes were classified into six coherent groups of differentially regulated genes, two of which, containing either NtcA-activated or NtcA-repressed genes, provided further insights into the function of NtcA–PipX complexes. The remaining four groups suggest the involvement of PipX in at least three NtcA-independent regulatory pathways. Our results pave the way to uncover new regulatory interactions and mechanisms in the control of gene expression in cyanobacteria.