Clock Mutant C44a Rescue via kaiC

Rescue of Clock Mutant C44a

• A long-period clock mutant, C44a (period = 44 hours), was transformed using a wild-type (WT) genomic DNA library for restoring regular circadian rhythms.
• The kaiC gene mutation was accountable for the altered period.
• Restoring a wild-type copy was able to correct the rhythm.
• Clock mutant C44a (44-hour period) was transformed using a plasmid DNA library with wild-type genomic fragments.
• Colonies were checked for restored wild-type 25-hour rhythms.
• Plasmid p44N, a rescued clone, was identified, which restored the normal period.
• The rescue of clock mutant C44a happens through WT kaiC expression, which restores circadian rhythms.
• Rescued clones displayed a 25-hour period, which is identical to wild-type.
• The correction shows the mutant had a recessive loss-of-function mutation in kaiC.
• Mutant C44a was transformed with a WT plasmid with a 44-hour period, proving kaiC mutation disrupted rhythmicity using restored clones having a normal 25-hour period.

The kaiABC Gene Cluster Map

• Shows the genetic map of the kaiABC gene cluster, which is crucial for circadian rhythms in cyanobacteria, confirming the three genes (kaiA, kaiB, kaiC) facilitate rhythm generation.
• The genomic DNA fragment (4.7 kb EcoRI segment) that rescued the mutant and was mapped by researchers.
• DNA sequencing revealed six open reading frames (ORFs).
• The three clock genes—kaiA, kaiB, and kaiC, form a single cluster.
• kaiA, kaiB, and kaiC genes are next to each other in the cyanobacteria genome.
• kaiC had the mutation in the mutant strain C44a.
• Southern blot analysis confirms that there is just one copy of this gene cluster in wild-type Synechococcus.
• The kaiABC gene cluster encodes essential circadian clock components and includes three clustered clock genes—kaiA, kaiB, and kaiC.
• A 4.7 kb DNA fragment containing clock genes was sequenced.

Amino Acid Sequences of Kai Proteins

• The amino acid sequences of KaiA, KaiB, and KaiC proteins are shown, highlighting key functional motifs.
• The predicted amino acid sequences of KaiA (284 residues), KaiB (102 residues), and KaiC (519 residues) and identified functional motifs in KaiC.
• KaiC functional motifs include Walker A & B motifs (ATP/GTP binding sites), catalytic glutamate residues (important for ATP hydrolysis), and DXXG motifs (GTP-binding proteins).
• KaiC is an ATP-binding protein, similar to clock proteins in other species; KaiA and KaiB do not have known enzymatic domains, but are crucial for rhythm regulation.
• KaiC has ATPase motifs and is structurally similar to clock proteins in other organisms, having Walker motifs (ATP-binding), suggesting an enzymatic role.
• Structural features suggest that KaiC plays a central role in the feedback loop.

Mapping of Clock Mutations

• Mutations found in kaiA, kaiB, and kaiC across various circadian rhythm mutants and confirms any of the three kai genes disrupt rhythms.
• The sequencing of the kai genes occurred in 19 clock mutants showing long/short periods or arrhythmic phenotypes with each mutant's exact mutation site mapped.
• 14 mutants had mutations in kaiC, 3 in kaiA, and 2 in kaiB; a single amino acid change in kaiC caused complete loss of rhythms.
• kaiABC is essential for the cyanobacterial clock.
• Mutations in kaiABC genes disrupt circadian rhythms (most mutations occurred in kaiC (14/19)), showing it is the key regulator of circadian rhythms.

Effects of Specific Mutations on Bioluminescence Rhythms

• Mutations in kaiC (A30a, B22a, C28a) show rhythmic bioluminescence (~25h period).
• CLAb mutation disrupts rhythms, proving its role in clock function.
• Mutants with specific kaiC mutations (A30a, B22a, C28a, CLAb) were assessed for bioluminescence rhythms in LL conditions.
• A30a, B22a, C28a mutants sustain ~25h rhythms, while CLAb mutant becomes arrhythmic.
• Most kaiC mutants retain rhythms (~25h), except CLAb, disruption from CLAb mutation proves kaiC's role.

KaiC Overexpression Disrupts Rhythmicity

• Overexpression of kaiC (via IPTG induction) abolishes rhythms, suggesting kaiC dosage is essential for clock function.
• kaiC was overexpressed in response to IPTG using the P_trc::kaiC inducible system and after IPTG bioluminescence rhythms was added.
• Wild-type rhythms (~25h) were maintained without IPTG, and KaiC overexpression (IPTG addition) abolishes oscillations.
• KaiC induction eliminates circadian rhythms, meaning KaiC dosage must be tightly regulated for clock function.

Timing of KaiC Overexpression Alters Phase

• The timing of kaiC overexpression (via IPTG) alters the phase of circadian rhythms.
• IPTG was added at different circadian phases with bioluminescence rhythms tracked to determine phase shifts, showing kaic levels influence phase timing.
• KaiC overexpression shifts the phase of circadian rhythms; KaiC supports to controls circadian phase.

Model of KaiC Regulation

• KaiC is regulated by transcription, translation, and protein interactions to maintain rhythmicity and has KaiA, KaiB, and KaiC interactions.
• KaiC translation maintains a tightly regulated clock in which KaiA, KaiB, and Kaic generate rhythmic feedback.
• KaiC regulation occurs at multiple levels (transcription, translation, feedback loops) and KaiA, KaiB, and KaiC form a post-translational oscillator (PTO).

Wild-Type (WT) Circadian Rhythms

• WT Synechococcus exhibits strong bioluminescence rhythms (~25h period) and compared against kai gene knockouts.
• WT Synechococcus was transformed with a lux bioluminescence reporter system that synchronized in darkness for 12h, then placed in continuous light (LL).
• Rhythmic bioluminescence was recorded over 96h, the WT cells show robust ~25h circadian rhythms, proving the reporter system is functional.
• WT Synechococcus showed normal 25h bioluminescence rhythms and strong circadian oscillations serve as a control for kai gene knockouts.

Deletion of kaiABC Causes Arrhythmicity

• Removing the entire kaiABC cluster abolishes rhythmicity, which confirms these genes are essential for the clock.
• The deletion of kaiABC genes via homologous recombination created a ΔkaiABC mutant strain in LL conditions.
• ΔkaiABC strain has no shows no oscillatory rhythm, only a gradual increase in bioluminescence.
• Growth remains normal → kaiABC is not required for survival, only for circadian function, while also disrupting the clock and causing arrhythmia.

Restoring kaiABC Rescues Rhythmicity

• Reintroducing kaiABC into the ΔkaiABC strain restores normal ~25h rhythms, proving these genes are both necessary and efficient for circadian function.
• The kaiABC cluster was reintroduced into the ΔkaiABC strain at a neutral site and proves kaiABC is necessary & efficient for rhythm generation.
• KaiABC is deemed the core clock and necessary and efficient for circadian rhythms.

Individual Kai Gene Knockouts

• Loss of kaiA, kaiB, or kaiC abolishes circadian rhythms, meaning each gene is required for proper circadian function because kaia, kaiB, kaiC were individually knocked out using targeted gene deletions.
• Fig. 2D (kaiA knockout), 2E (kaiB knockout), 2F (kaiC knockout) are Arrhythmic, individually proving essentialness for oscillation and is why these factors are removed for deletion.

Bioluminescence Rhythms from kai Promoters

• The use of lux reporters shows the rhythmic transcription of kaiA, kaiB, and kaiC where expression is arrhythmic, suggesting post-transcriptional regulation.
• Fusion constructs (USRkaiA::lux, USRkaiB::lux, USRkaiC::lux) were created by linking kai promoters to a lux bioluminescence reporter and used for bioluminescence rhythms measurements under LL conditions.
• kaiA & kaiB show robust oscillations (~25h period), while kaiC lacks oscillations implying it's not transcriptional but post-transcriptional via lux fusions.
• kaiA and kaiB exhibit rhythmic oscillation, but kaiC suggesting post-transcriptional.

Bioluminescence from kaiABC Reporter

• The entire kaiABC operon exhibits rhythmic expression, which suggests kaiA/kaiB drive transcriptional rhythms, while kaiC is regulated post-transcriptionally.
• Fusing luxAB created A kaiABC::lux reporter to the kaiABC operon and found that kaiABC operon expression is rhythmic and in fusion with rhythms.

Bioluminescence from psbAl Reporter

• psbAI expression oscillates (~25h period) with a psbAI promoter fused to lux to track circadian oscillations.
• The psbAI control gene exhibits strong circadian rhythms with strong oscillations (~25h period) similar to kaiA and kaiB confirming the validity of the reporter.

Northern Blot of kai Transcripts

• kaiC transcript is larger (~2.3 kb), supporting differential processed and post-transcriptional regulation.
• RNA extraction was completed from WT Synechococcus, and Northern blot analysis was performed using kaiA, kaiB, kaiC-specific probes with kai transcript measurements indicating post-transcriptional regulation, suggesting that Northern blots indicate alternative regulation.

Circadian Expression of kaiA and kaiC mRNA

• Northern blot analysis confirmed kaiA and kaiC mRNA accumulate rhythmically (~25h period) with circadian regulation at the transcriptional level.
• Experiments were completed on northern blots of kaiA and kaiC to detect mRNA rhythms.
• Measurements were completed for 48h in LL conditions, indicating support for alternative circadian regulation.