A Cyanobacterial Circadian Timing Mechanism

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A Cyanobacterial Circadian Timing Mechanism

• J.L. Ditty, S.B. Williams, and S.S. Golden
• Presented by Angela Baioni

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Introduction

• Majority of organisms subjected to daily
  fluctuations in light and temperature.
• Evolution of mechanisms to adjust to/anticipate
  daily environmental changes
• Several mechanisms to study circadian phenomena
  S. elongatus PCC 7942
• Many players involved in every aspect of the
  clock
• Many questions still unanswered

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Cyanobacteria

• Photoautotrophic prokaryotes
• Genetic lineage among oldest on Earth
• Oxygenic photosynthesis originated here
• Remarkable genetic diversity
• Morphological diversity
• Reside in nearly every habitat sunlight
  penetrates
• Wide range of growth rates and metabolic
  activities
• Participation in symbiotic associations

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• What biological properties have helped define
  cyanobacteria?
• Speculation that presence of circadian clock
  genes and a functional circadian clock are
  also cyanobacterial characteristics.

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Circadian Clock

• Endogenous cellular mechanism
• Temporal organization
• General models involve 3 basic elements
• -circadian oscillator
• -input pathways
• -ouput pathways

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Fig 1
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Characteristics of Clock

• 1.) Circadian rhythms persist under constant
  environmental conditions
• Free-run
• Period

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• 2.) Circadian systems sensitive to environmental
  stimulus to achieve entrainment.
• 3.) Timing mechanism maintains periodicity
  through temperature compensation.
• Intrinsic periods vary depending upon
  environmental conditions.
• Aschoffs Rule

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Fig 2
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Cyanobacterial Circadian Clock

• 1980s evidence of timing of onset of nitrogen
  fixation BUT circadian regulation restricted to
  eukaryotic organisms
• 1st convincing evidence in 1989-90 with Sweeney
  and Borgese WH 7803
• Leading up to development of S. elongatus PCC 7942

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Model System PCC 7942

• Cannot fix nitrogen
• Selective growth advantage owing to its clock
• Understanding of how clock controls certain
  processes is very limited.
• Models emerging that can be used to study
  underlying mechanisms.

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• Extremely malleable to various techniques
• Clock comprised of at least 3 genes kaiA, B,
  and C

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• At least 800 of 2700 genes expressed rhythmically
• Gene expression patterns can be categorized b/c
  differences are promoter specific
• Class I (most pervasive form) peak near
  subjective dusk
• Class II antiphase (peak at subjective dawn)

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Fig 4
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Kai A, B, and C

• Kai A 855bp, encodes 32.6 kD protein
• Kai B 309bp, encodes 11.4 kD protein
• Kai C is 1560 bp, encodes 58 kD protein

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• Kai C 2 Walkers A motifs, 2 Walkers B motifs,
  and 2 catalytic sites present in other
  ATP-binding proteins
• DXXG motifs conserved in GTPase
• Similarities between C1 and C2 with one of ATP
  motifs
• Kai A and Kai B have no recognizable motifs
• C1 and C2 have distinct functions, bind 1 ATP
  (2/Kai C)

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• Kai C can autophosphorylate when incubated with
  ATP in vitro
• Occurs at serine and/or threonine residues
• ATP binding ability of Kai C important for
  timekeeping in oscillator
• Kai A and B have no autokinase activity
• Play role in autophosphorylation of Kai C

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Fig 6
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• 2 forms of Kai A exist long and short

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• Kai A B opposing modulators for Kai C
  autophosphorylation
• Kai A C remains phosphorylated
• Kai A B C dephosphorylates but at reduced
  rate than Kai C alone
• Kai B C no effect
• Kai A essential for in vivo phosphorylation of
  Kai C

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Fig 8
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• Autokinase activity of Kai C is rhythmic
• Kai proteins present in large complexes that
  changed stoichiometry over the cycle
• Kai A C localized in cytosol
• Kai B is localized to the membrane
• Kai C forms higher order complex with itself
• Hexameric form dependent upon presence of ATP
• ATP binding essential for hexamerization
  process is reversible
• High interactions of Kai C indicate it may be
  driving force for circadian rhythms

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Fig 9
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Transcriptional Control

• Negative feedback loops essential for regulation
• In eukaryotic systems, have positive elements and
  negative elements
• Kai C protein required for wt levels of
  expression in kaiBC promoter
• Kai A a positive activator
• Cooperative effort between Kai A and C?

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Input CIKA, LPDA, Pex

• CikA key player
• 3 motifs GAF, HPK, and RR

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• LdpA affects input in different way than CikA
• Contains 2 Fe-S centers
• Pex is suppressor of 22 hr period
• 447bp and encodes 17-kD protein with no obvious
  functional motifs
• Loss of pex greatly increases expression of kaiA
• No specific role in input pathway can be assigned

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Output SasA, CPMA, and Group 2 Sigma Factors

• SasA N-terminus strongly resembles Kai B protein
• Full length SasA interacts with itself and Kai C
• Associates closely with Kai oscillator complex
• Important for rhythms of transcription from
  downstream genes

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• Also acts on input pathway
• Strongest evidence Kai C SasA
  autophosphorylation of SasA but has no effect on
  Kai C autophosphorylation
• Sigma factors
• Cyanobacteria have multiple, closely related
  sigma factors not essential for growth Group 2
• Working model states transcription apparatus
  oscillates in circadian manner

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• CpmA gene
• Mutations in expression did affect point timing
  but overall organismal timing is still maintained

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Discussion

• Kai genes confirmed to be essential components
• Genomes sequenced and all had at least one kai
  gene
• Kai A plays essential role in rhythm generation
  but N-terminal RR absent in many species
• Not surprising due to evolutionary vastness

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• Main requirement is ability to receive
  environmental stimuli and dock the Kai complex,
  inducing conformational change
• Evidence suggests that kaiBC operon is nearly 2
  billion years old
• Seems likely that common ancestor between
  cyanobacteria and plastids had kai genes
• But where are the kai gene homologs in plastid
  lineage?

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Model for PCC 7942 timekeeping
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Thank you ..