Synechococcus: KaiABC Gene Cluster

• Genomic DNA library from wild-type (WT) Synechococcus was used to transform a long-period clock mutant (C44a) to restore normal circadian rhythms.
• The kaiC gene mutation in C44a caused the altered period, and restoring a wild-type copy corrected the rhythm.
• Rescued clones displayed a 25-hour period, matching wild-type Synechococcus.
• Loss-of-function mutation in kaiC is recessive.

Map of the kaiABC Gene Cluster

• The kaiABC gene cluster, is vital for circadian rhythms in cyanobacteria.
• The 4.7 kb EcoRI genomic DNA fragment rescued the mutant.
• The 4.7 kb fragment contains three clock genes (kaiA, kaiB, kaiC) comprising a gene cluster.
• The kaiC gene had the mutation in the mutant strain C44a.
• No other copies of the kaiABC gene cluster exist in wild-type Synechococcus.

Deduced Amino Acid Sequences of Kai Proteins

• KaiC sequence contains Walker A and B motifs which are ATP/GTP binding sites.
• It also contains Catalytic glutamate residues and DXXG motifs.
• Predicted amino acid sequences: KaiA (284 residues), KaiB (102 residues), and KaiC (519 residues) were analyzed.
• KaiC structural features indicate it plays a main role in the feedback loop
• KaiA and KaiB lack known enzymatic domains. KaiA and KaiB are still critical for rhythm regulation.
• KaiC is an ATP-binding protein, like clock proteins across species.

Mapping of Clock Mutations

• The kai genes were sequenced in 19 clock mutants exhibiting long/short periods or arrhythmia.
• kaiC is likely the most important regulator as 14/19 mutants had kaiC mutations.
• Mutation of a single amino acid in kaiC caused a complete loss of rhythms.
• Three mutants had mutations in kaiA, and two had mutations in kaiB.

Wild-Type (WT) Circadian Rhythms

• Wild-type Synechococcus exhibits strong bioluminescence rhythms with an approximate 25-hour period measured using a lux bioluminescence reporter system.
• The lux bioluminescence reporter system demonstrates functionality in cells synchronized in darkness for 12 hours, then placed in continuous light (LL).

Deletion of kaiABC (ΔkaiABC) Causes Arrhythmicity

• The kaiABC cluster's removal abolishes rhythmicity.
• The kaiABC genes were deleted using homologous recombination.
• ΔkaiABC strain only shows a gradual increase in bioluminescence with no rhythm.
• Deletion of kaiABC does not impact growth. Only for circadian function.

Restoring kaiABC Rescues Rhythmicity

• Reintroducing kaiABC into the ΔkaiABC strain at a neutral site restores normal rhythms.
• Shows a wild type 25-hour rhythm.
• kaiABC is necessary and sufficient for circadian function.

Individual Kai Gene Knockouts

• Deleting any single kai gene (kaiA, kaiB, kaiC) abolishes circadian rhythms.
• Loss of kaiA, kaiB, or kaiC eliminates circadian rhythms.
• Targeted gene deletions were used to individually knockout kaiA, kaiB, kaiC.

Bioluminescence Rhythms from kai Promoters

• kaiA and kaiB exhibit robust oscillations (≈25h period).
• kaiC does not show oscillations suggesting post-transcriptional regulation using lux reporters.
• lux reporters reveal rhythmic expression of kaiA, kaiB, and kaiC.

Bioluminescence from kaiABC::lux Reporter

• The entire kaiABC operon exhibits rhythmic expression measured in continuous light (LL) using a kaiABC::lux reporter.
• kaiA and kaiB drive transcriptional rhythms, while kaiC is regulated post-transcriptionally
• oscilliations last are approximately 25 hours

Bioluminescence from psbAI::lux Reporter

• psbAI (control gene) shows strong circadian rhythms similar to kaiA and kaiB with approxiamtely 25 hour period using a psbAI::lux fusion to track circadian oscillations.
• Confirms the validity of the bioluminescence system.

Northern Blot of kai Transcripts

• kaiA, kaiB, and kaiC are all transcribed shown through Northern blot analysis using kaiA, kaiB, kaiC-specific probes.
• kaiC transcript is larger (~2.3 kb), supporting post-transcriptional regulation.
• RNA was extracted from wild type Synechococcus.

Circadian Expression of kaiA and kaiC mRNA

• Northern blot analysis performed on RNA over 48 hours.
• kaiA and kaiC mRNA levels oscillate (~25h period).
• Supports circadian regulation at the transcriptional level.

Effects of Specific Mutations on Bioluminescence Rhythms

• Mutations A30a, B22a, C28a in kaiC still show rhythmic bioluminescence (~25 hour period).
• The CLAb mutation disrupts rhythms disrupting clock integrity.
• Rhythm data was gathered by measuring bioluminescence rhythms in kaiC mutants under LL conditions.

kaiC Overexpression Disrupts Rhythmicity

• Overexpression of kaiC (through IPTG induction) abolishes rhythms, as measured by bioluminescence.
• kaiC dosage must be tightly regulated for clock function as overtime of kaiC disrupts rhythmicity.
• P_trc::kaiC inducible system used to overexpress kaiC with IPTG.

Timing of kaiC Overexpression Alters Phase

• Timing of kaiC overexpression (via IPTG) alters circadian rhythms.
• Rhythm data was measured by recording bioluminesence after adding IPTG at different times and phases.
• kaiC levels influence phase timing.

Proposed Model of kaiC Regulation in the Circadian Clock

• Conceptual model summarizing findings on kaiA, kaiB, and kaiC interactions.
• kaiC translation is tightly regulated to maintain the clock. Interactions between KaiA, KaiB, and KaiC generate rhythmic feedback.
• kaiC is regulated by transcription, translation, and protein interactions to maintain rhythmicity.
• KaiA, KaiB, and KaiC form a Post-translational oscillator (PTO).