A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock

Abstract

Two-component systems (TCS) that employ histidine kinases (HK) and response regulators (RR) are critical mediators of cellular signaling in bacteria. In the model cyanobacterium Synechococcus elongatus PCC 7942, TCSs control global rhythms of transcription that reflect an integration of time information from the circadian clock with a variety of cellular and environmental inputs. The HK CikA and the SasA/RpaA TCS transduce time information from the circadian oscillator to modulate downstream cellular processes. Despite immense progress in understanding of the circadian clock itself, many of the connections between the clock and other cellular signaling systems have remained enigmatic. To narrow the search for additional TCS components that connect to the clock, we utilized direct-coupling analysis (DCA), a statistical analysis of covariant residues among related amino acid sequences, to infer coevolution of new and known clock TCS components. DCA revealed a high degree of interaction specificity between SasA and CikA with RpaA, as expected, but also with the phosphate-responsive response regulator SphR. Coevolutionary analysis also predicted strong specificity between RpaA and a previously undescribed kinase, HK0480 (herein CikB). A knockout of the gene for CikB (cikB) in a sasA cikA null background eliminated the RpaA phosphorylation and RpaA-controlled transcription that is otherwise present in that background and suppressed cell elongation, supporting the notion that CikB is an interactor with RpaA and the clock network. This study demonstrates the power of DCA to identify subnetworks and key interactions in signaling pathways and of combinatorial mutagenesis to explore the phenotypic consequences. Such a combined strategy is broadly applicable to other prokaryotic systems. IMPORTANCESignaling networks are complex and extensive, comprising multiple integrated pathways that respond to cellular and environmental cues. A TCS interaction model, based on DCA, independently confirmed known interactions and revealed a core set of subnetworks within the larger HK-RR set. We validated high-scoring candidate proteins via combinatorial genetics, demonstrating that DCA can be utilized to reduce the search space of complex protein networks and to infer undiscovered specific interactions for signaling proteins in vivo. Significantly, new interactions that link circadian response to cell division and fitness in a light/dark cycle were uncovered. The combined analysis also uncovered a more basic core clock, illustrating the synergy and applicability of a combined computational and genetic approach for investigating prokaryotic signaling networks. Bacterial two-component systems (TCS) are ubiquitous signaling modules consisting of at least one histidine kinase (HK) and one response regulator (RR) (1), which transduce signals via transfer of a phosphate group from the HK to a receiver domain on the RR. Over the years, studies have expanded the common paradigm of TCS signaling via phosphotransfer between single HK-RR pa...

Designing bacterial signaling interactions with coevolutionary landscapes

“…Coevolutionary landscapes can also be used to identify TCS partner interactions within an organism [ 18 , 41 ], i.e., which HKs and RRs preferentially interact. These approaches are consistent with earlier approaches that used information-based scores [ 16 , 19 , 42 ].…”

Guiding the design of bacterial signaling interactions using a coevolutionary landscape

“…It has previously been shown that coevolutionary landscapes can also be used to identify TCS partner interactions (Bitbol, Dwyer et al, 2016, i.e., which HKs and RRs preferentially interact. These approaches are consistent with earlier approaches that used information-based scores (Boyd, Cheng et al, 2016, Cheng et al, 2014, Procaccini, Lunt et al, 2011.…”

Energy drives the dynamic localization of cyanobacterial nitrogen regulators during diurnal cycles

“…They are the simplest organisms known to have a circadian clock, extensively studied in the model cyanobacterium Synechococcus elongatus PCC7942 (hereafter S. elongatus) (Shultzaberger et al, 2015), where it comprises a core oscillator which relay timing information to the output pathway, generating genome-wide transcriptional oscillations (Liu et al, 1995). Cross-talk with other two-component regulators allows integration of additional environmental signals (Markson et al, 2013;Espinosa et al, 2015;Boyd et al, 2016). In S. elongatus, diurnal fluctuations appear to be less pronounced at the protein level than at the transcript levels (Guerreiro et al, 2014).…”