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.

Profiling in sport using momentum and perturbations

Examining a team’s performance from a physical point of view their momentum might indicate unexpected turning points in defeat or success. Physicists describe this value as to require some effort to be started, but also that it is relatively easy to keep it going once a sufficient level is reached (Reed and Hughes, 2006). Unlike football, rugby, handball and many more sports, a regular volleyball match is not limited by time but by points that need to be gathered. Every minute more than one point is won by either one team or the other. That means a series of successive points enlarges the gap between the teams making it more and more difficult to catch up with the leading one. This concept of gathering momentum, or the reverse in a performance, can give the coaches, athletes and sports scientists further insights into winning and losing performances. Momentum investigations also contain dependencies between performances or questions if future performances are reliant upon past streaks. Squash and volleyball share the characteristic of being played up to a certain amount of points. Squash was examined according to the momentum of players by Hughes et al. (2006). The initial aim was to expand normative profiles of elite squash players using momentum graphs of winners and errors to explore ‘turning points’ in a performance. Dynamic systems theory has enabled the definition of perturbations in sports exhibiting rhythms (Hughes et al., 2000; McGarry et al., 2002; Murray et al., 2008), and how players and teams cause these disruptions of rhythm can inform on the way they play, these techniques also contribute to profiling methods. Together with the analysis of one’s own performance it is essential to have an understanding of your oppositions’ tactical strengths and weaknesses. By modelling the oppositions’ performance it is possible to predict certain outcomes and patterns, and therefore intervene or change tactics before the critical incident occurs. The modelling of competitive sport is an informative analytic technique as it directs the attention of the modeller to the critical aspects of data that delineate successful performance (McGarry & Franks, 1996). Using tactical performance profiles to pull out and visualise these critical aspects of performance, players can build justified and sophisticated tactical plans. The area is discussed and reviewed, critically appraising the research completed in this element of Performance Analysis.

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.