1 / 34

Circadian Rhythms in Synechococcus elongatus

Circadian Rhythms in Synechococcus elongatus. John Buchner jbuchner@ucsd.edu. Earth’s Rotation is a Constant. What Organisms Have Circadian Rhythms?. Working Definition of Circadian Systems. ≈24 cycle Entrainment by External Signal Light and Dark Warmer and Colder

blaine
Download Presentation

Circadian Rhythms in Synechococcus elongatus

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Circadian Rhythms in Synechococcus elongatus John Buchner jbuchner@ucsd.edu

  2. Earth’s Rotation is a Constant

  3. What Organisms Have Circadian Rhythms?

  4. Working Definition of Circadian Systems • ≈24 cycle • Entrainment by External Signal • Light and Dark • Warmer and Colder • Maintained in the Absence of External Signal • Temperature Compensated

  5. Functions of Clocks • Control of Organism’s • Gene Expression • Metabolism • Activity Cycles • Optimize Activity to Environment • Out of Sync Clocks = Less Fitness

  6. Synechococcus elongatus PCC 7942 • Single-celled cyanobacterium • Photosynthesis is its only means of growth • Small genome (approx 2800 genes) • Takes up foreign DNA naturally • Easy to knock genes out, increase expression of genes • Robust circadian rhythms of gene expression

  7. Cyanobacterial Circadian Rhythms • Nitrogen fixation • Photosynthesis • Amino acid uptake • Gene expression • Timing of cytokinesis • Confers fitness to progeny

  8. luxA luxB Synechococcus elongatus PCC 7942

  9. How to Insert Reporters:

  10. How Many Genes Are Under Circadian Control? • 10% Detectable Luminescence (>20,000 Colonies Screened, Closer to 30000?) • Colonies separated into “Brights” (≥130 CPM) and “Dims” (<130-13 CPM) • Bioluminescent Rhythms Detected in • 798 Selected Brights (“Almost every bright colony...”) • 40 Selected Dims • Controls: • Southern Blot Indicates Randomness of Insertions • Northern Blot of Known Genes • Known Rhythmic Promoters also had Bioluminescent Rhythms • Conclusion: All genes in S. elongatus are Rhythmic • Assumptions: • The Non-Trackable Clones are Rhythmic • Insertions are really random, and low chance of duplication Liu et. al. 1995

  11. Bioluminescence Assay 5,000 Bioluminescence (cps) 0 0 12 24 36 48 60 72 84 96 Hours in LL Analysis of rhythms Inoculation of reporter strains Bedding test strains on agar pads Real-time bioluminescence detection

  12. light 120 Promoter::luciferase fusions as reporter 80 40 0 Circadian Rhythms of Gene Expression period phase Relative bioluminescence amplitude 0 24 48 72 96 120 Time (h)

  13. kaiA, kaiBC Required for Functional Clock k a i A k a i B k a i C - 1 k b

  14. KaiC is a Autophosphatase, Autokinase, ATPase P P ATP ADP + P P P ATP ADP + P ATP ADP + P ATP ADP + P

  15. … but Without KaiAB, KaiC No Cycling Occurs ATP ADP + P ATP ADP + P ATP ADP + P ATP ADP + P

  16. KaiA Stimulates Phosphorylation, ATPase Activities KaiA P P ATP ADP + P KaiA P P ATP ADP + P ATP ADP + P KaiA ATP ADP + P

  17. KaiB Antagonizes KaiA Activites KaiB KaiA P P ATP ADP + P KaiB KaiA P P ATP ADP + P ATP ADP + P KaiA KaiB ATP ADP + P

  18. Invitro Oscillation

  19. KaiB KaiC KaiC The Central Oscillator is Not the Clock ADP ATP KaiA KaiA P P P P P

  20. Input pathways provide synchronization with the environment A central oscillator keeps 24 h time

  21. KaiB KaiC KaiC Expansion out from the Central Oscillator KaiA redox state Pex LdpA CikA Q ADP ATP KaiA KaiA P P P P P

  22. GAF Rec HPK P ATP ADP PsR Features of CikA Histidine Protein Kinase Activates HPK Q Pseudo Receiver

  23. cikA PsR GAF HPK CikA is essential for environmental input wild type

  24. CikA Mutants are Defective in Cell Division WT cikA • Poor localization of FtsZ ring • Is this defect related to CikA clock function? • What else causes cell elongation? • KaiA Over-expression • KaiB Deletion • Stimulation of KaiC ATPase activity

  25. KaiB KaiC KaiC LdpA, Pex KaiA redox state Pex LdpA CikA Q ADP ATP KaiA KaiA P P P P P

  26. Input pathways provide synchronization with the environment A central oscillator keeps 24 h time Output pathways transmit temporal information to clock-controlled behaviors CikA KaiA, KaiB, KaiC SasA

  27. KaiB KaiC KaiC Clock Output: ADP ATP KaiA KaiA KaiA P P redox state Pex P P SasA LdpA CikA P P ATP Q RpaA ADP LabA Gene Regulation, Chromosome Compaction, Cell Division

  28. Phenotypes of sasA and rpaA nulls • Lower Levels Gene Expression • Decreased Amplitude • Short Period • Severe Growth Defect in LD cycles • Clock fails to regulate Cell Division

  29. The clock gates cell division 6 5 4 Relative Frequency 3 2 1 0 0 5 1 0 1 5 2 0 2 5 C i r c a d i a n P h a s e [ h ]

  30. Why is Cell Division Stopped? • Most Cellular functions continue • DNA Replication • Transcription/Translation • Metabolism • Perhaps the Gate Protects Something • The Clock • Specific Metabolic Machinery • Chromosome

  31. Chromosome Compaction in S. elongatus 0 4 8 12 16 20 24 Time (hours)

  32. In Summary • S. elongatus has a functional Circadian Clock • KaiABC Comprise the Central Oscillator • Loss of any = no Circadian Clock • Oscillations can be Reconstituted in vitro • Time Measured by Redox State (Not Light!) • Outputs Include • Gene Expression Rhythm • Chromosome Compaction

More Related