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

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 informa...

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Veröffentlicht in:Journal of bacteriology 2016-09, Vol.198 (18), p.2439-2447
Hauptverfasser: Boyd, Joseph S, Cheng, Ryan R, Paddock, Mark L, Sancar, Cigdem, Morcos, Faruck, Golden, Susan S
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container_end_page 2447
container_issue 18
container_start_page 2439
container_title Journal of bacteriology
container_volume 198
creator Boyd, Joseph S
Cheng, Ryan R
Paddock, Mark L
Sancar, Cigdem
Morcos, Faruck
Golden, Susan S
description 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. Signaling 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 uncove
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J.</contributor><creatorcontrib>Boyd, Joseph S ; Cheng, Ryan R ; Paddock, Mark L ; Sancar, Cigdem ; Morcos, Faruck ; Golden, Susan S ; Christie, P. J.</creatorcontrib><description>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. Signaling 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. 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All Rights Reserved.</rights><rights>Copyright American Society for Microbiology Sep 2016</rights><rights>Copyright © 2016, American Society for Microbiology. 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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. Signaling 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. 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J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock</atitle><jtitle>Journal of bacteriology</jtitle><addtitle>J Bacteriol</addtitle><date>2016-09-15</date><risdate>2016</risdate><volume>198</volume><issue>18</issue><spage>2439</spage><epage>2447</epage><pages>2439-2447</pages><issn>0021-9193</issn><eissn>1098-5530</eissn><coden>JOBAAY</coden><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. 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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. Signaling 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.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>27381914</pmid><doi>10.1128/JB.00235-16</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record>
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subjects Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Bacteriology
Circadian Clocks - physiology
Circadian rhythm
Computer Simulation
Cyanobacteria
Evolution, Molecular
Gene Expression Regulation, Bacterial - physiology
Genetics
Gram-negative bacteria
Kinases
Mutagenesis
Mutation
Phosphorylation
Signal transduction
Signal Transduction - physiology
Synechococcus - genetics
Synechococcus - metabolism
Synechococcus elongatus
title A Combined Computational and Genetic Approach Uncovers Network Interactions of the Cyanobacterial Circadian Clock
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