An allele of the
            <i>crm</i>
            gene blocks cyanobacterial circadian rhythms

Boyd, J. Morton; Bordowitz, Juliana R.; Bree, Anna C.; Golden, Susan S.

doi:10.1073/pnas.1312793110

Cited by 24 publications

(27 citation statements)

References 29 publications

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“…Suspected sister colonies also exhibited similar circadian phenotypes. The sixth genome did not contain a mutation in sasA, but rather had a point mutation in the intergenic region upstream of the circadiancontrolled transcriptional regulator rpaA and downstream of the crm gene (12). This mutant displayed a further disruption in bioluminescence amplitude relative to the cikA-null strain, approaching arrhythmia.…”

Section: Resultsmentioning

confidence: 99%

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Single mutations in sasA enable a simpler Δ cikA gene network architecture with equivalent circadian properties

Shultzaberger

Boyd

Katsuki

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The circadian input kinase of the cyanobacterium Synechococcus elongatus PCC 7942 (CikA) is important both for synchronizing circadian rhythms with external environmental cycles and for transferring temporal information between the oscillator and the global transcriptional regulator RpaA (regulator of phycobilisome-associated A). KOs of cikA result in one of the most severely altered but still rhythmic circadian phenotypes observed. We chemically mutagenized a cikA-null S. elongatus strain and screened for second-site suppressor mutations that could restore normal circadian rhythms. We identified two independent mutations in the Synechococcus adaptive sensor A (sasA) gene that produce nearly WT rhythms of gene expression, likely because they compensate for the loss of CikA on the temporal phosphorylation of RpaA. Additionally, these mutations restore the ability to reset the clock after a short dark pulse through an output-independent pathway, suggesting that SasA can influence entrainment through direct interactions with KaiC, a property previously unattributed to it. These experiments question the evolutionary advantage of integrating CikA into the cyanobacterial clock, challenge the conventional construct of separable input and output pathways, and show how easily the cell can adapt to restore phenotype in a severely compromised genetic network.gene network | cyanobacteria | evolution S econd-site suppressor mutagenesis screens have been useful in untangling the genetic components and pathways that underlie complex cellular phenotypes (1). By understanding how mutations in one gene can compensate for changes in another, we can infer how those genes interact with each other and with their encompassing genetic network. We used this approach to better understand the genetic determination of circadian rhythms in the cyanobacterium Synechococcus elongatus PCC 7942. Because the core clock genes in this species exist as single copies, suppressing mutations are not buffered by paralogs, making S. elongatus ideal for this type of mutagenic assay. Approximately 15 genes have been identified that influence the circadian phenotype to various degrees, the most important of which are the kai genes: kaiA, kaiB, and kaiC. In cyanobacteria, time is kept through the central oscillator protein KaiC, whose phosphorylation state, conformation, and ATPase activity, which are mediated by interactions with KaiA and KaiB, cycle within a 24-h period (2-4). The temporal phosphorylation of KaiC can be reconstituted in vitro by simply combining the three Kai proteins and ATP, suggesting that time is kept primarily through a posttranslational mechanism (5). Although the Kai oscillator functions relatively robustly in isolation, additional proteins are necessary to synchronize the oscillations with the environment (entrainment) through the detection of cellular and environmental cues (input pathway) and to transfer temporal information from the oscillator to circadian-dependent transcriptional regulators (output pathway). One of these prot...

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Section: Resultsmentioning

confidence: 99%

“…For immunoblotting, SDS/PAGE gels were prepared with Phos-tag reagent (Wako Chemicals USA) and whole-cell protein extracts were prepared as described previously (12). RpaA and KaiC were detected using appropriate antisera as described by Boyd et al (12) and Dong et al (34).…”

Section: Methodsmentioning

confidence: 99%

Single mutations in sasA enable a simpler Δ cikA gene network architecture with equivalent circadian properties

Shultzaberger

Boyd

Katsuki

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…All ΔrpaA mutants were constructed by transformation in a WT S. elongatus background with plasmid pAM4420 (25) and were validated by PCR. For all experiments, precultures were first prepared in 100 mL of fresh BG-11 medium as in Diamond et al (19).…”

Section: Methodsmentioning

confidence: 99%

“…Although it is known that ΔrpaA strains do not grow under LD conditions (19,25), the nature of the defect has not been characterized. We initially examined changes in cell viability and whole-cell absorbance as cultures entered the dark.…”

Section: Darkness Initiates Pigmentation Changes and Rapid Cell Deathmentioning

confidence: 99%

“…This temporal activity pattern, and the fact that RpaA directly binds and activates nighttime metabolism genes (20,24), suggests that it also plays an important role in metabolic control at night. Although the transcriptional targets of RpaA have been identified, and it is clear that LD conditions are deleterious to rpaA-null mutants (25), the metabolic and physiological changes that attenuate growth under LD conditions have not been explored.…”

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Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

Diamond

Rubin

Shultzaberger

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Cyanobacteria evolved a robust circadian clock, which has a profound influence on fitness and metabolism under daily lightdark (LD) cycles. In the model cyanobacterium Synechococcus elongatus PCC 7942, a functional clock is not required for diurnal growth, but mutants defective for the response regulator that mediates transcriptional rhythms in the wild-type, regulator of phycobilisome association A (RpaA), cannot be cultured under LD conditions. We found that rpaA-null mutants are inviable after several hours in the dark and compared the metabolomes of wild-type and rpaA-null strains to identify the source of lethality. Here, we show that the wild-type metabolome is very stable throughout the night, and this stability is lost in the absence of RpaA. Additionally, an rpaA mutant accumulates excessive reactive oxygen species (ROS) during the day and is unable to clear it during the night. The rpaA-null metabolome indicates that these cells are reductant-starved in the dark, likely because enzymes of the primary nighttime NADPH-producing pathway are direct targets of RpaA. Because NADPH is required for processes that detoxify ROS, conditional LD lethality likely results from inability of the mutant to activate reductant-requiring pathways that detoxify ROS when photosynthesis is not active. We identified second-site mutations and growth conditions that suppress LD lethality in the mutant background that support these conclusions. These results provide a mechanistic explanation as to why rpaA-null mutants die in the dark, further connect the clock to metabolism under diurnal growth, and indicate that RpaA likely has important unidentified functions during the day.C yanobacteria are both key agents of global carbon and nitrogen cycles and promising platforms for renewable chemicals, fuels, and nutraceuticals (1-3). Understanding the control mechanisms that govern the flow of carbon and nitrogen through these organisms is crucial for predicting their behavior in natural environments as well as for improving engineering strategies. Although the basic pathways for carbon and nitrogen metabolism, and their regulation, are well understood in heterotrophic bacteria, cyanobacteria exhibit important deviations in these core metabolic pathways (4-7). Additionally, metabolic control mechanisms in cyanobacteria evolved to be compatible with photoautotrophic metabolism and the dramatic shifts that are imposed on those pathways by predictable daily light-dark (LD) cycles. Examples include enzymatic activity that responds to light-dependent cellular redox changes (8-11); the preference for NADPH, the reductant produced by the photochemical reactions, over NADH by many biosynthetic enzymes (12, 13); and a circadian clock that drives 24-h transcriptional rhythms in most genes (14-16).A daily LD cycle presents a strong metabolic driver for the photosynthetic cyanobacteria, but a circadian clock also imposes daily cycles in transcription and redox regulatory systems (17, 18). Circadian measurements historically have been performed in...

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The Kai-Protein Clock—Keeping Track of Cyanobacteria’s Daily Life

Snijder

Axmann

2019

Subcellular Biochemistry

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An allele of the crm gene blocks cyanobacterial circadian rhythms

Cited by 24 publications

References 29 publications

Single mutations in sasA enable a simpler Δ cikA gene network architecture with equivalent circadian properties

Single mutations in sasA enable a simpler Δ cikA gene network architecture with equivalent circadian properties

Redox crisis underlies conditional light–dark lethality in cyanobacterial mutants that lack the circadian regulator, RpaA

The Kai-Protein Clock—Keeping Track of Cyanobacteria’s Daily Life

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