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. 2013 Mar;195(6):1276-84.
doi: 10.1128/JB.02067-12. Epub 2013 Jan 11.

Genome-wide and Heterocyst-Specific Circadian Gene Expression in the Filamentous Cyanobacterium Anabaena Sp. Strain PCC 7120

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Free PMC article

Genome-wide and Heterocyst-Specific Circadian Gene Expression in the Filamentous Cyanobacterium Anabaena Sp. Strain PCC 7120

Hiroko Kushige et al. J Bacteriol. .
Free PMC article

Abstract

The filamentous, heterocystous cyanobacterium Anabaena sp. strain PCC 7120 is one of the simplest multicellular organisms that show both morphological pattern formation with cell differentiation (heterocyst formation) and circadian rhythms. Therefore, it potentially provides an excellent model in which to analyze the relationship between circadian functions and multicellularity. However, detailed cyanobacterial circadian regulation has been intensively analyzed only in the unicellular species Synechococcus elongatus. In contrast to the highest-amplitude cycle in Synechococcus, we found that none of the kai genes in Anabaena showed high-amplitude expression rhythms. Nevertheless, ~80 clock-controlled genes were identified. We constructed luciferase reporter strains to monitor the expression of some high-amplitude genes. The bioluminescence rhythms satisfied the three criteria for circadian oscillations and were nullified by genetic disruption of the kai gene cluster. In heterocysts, in which photosystem II is turned off, the metabolic and redox states are different from those in vegetative cells, although these conditions are thought to be important for circadian entrainment and timekeeping processes. Here, we demonstrate that circadian regulation is active in heterocysts, as shown by the finding that heterocyst-specific genes, such as all1427 and hesAB, are expressed in a robust circadian fashion exclusively without combined nitrogen.

Figures

Fig 1
Fig 1
Expression of Anabaena kai genes is not robustly rhythmic under LL conditions. (A) Total RNAs were extracted from Anabaena cells that had been collected every 4 h from a continuous culture under LL after two LD cycles in the presence of sodium nitrate. They were subjected to Northern hybridization analysis using the extracted RNAs (1.0 μg) of each Anabaena kaiA, kaiB, kaiC, or kaiBL sequence as probes. As a reference, the high-amplitude kaiBC mRNA profile in Synechococcus is also shown. (B) Densitometric analysis of kai gene expression under LL, obtained with DNA microarray experiments. The dots represent the signals from two independent experiments, and the solid lines indicate average expression levels. The numbers on the ordinate indicate the relative expression levels.
Fig 2
Fig 2
Genome-wide circadian transcription profiles under LL conditions. (A) Variations in the oscillatory indices, amplitude (defined as standard deviation [SD] normalized to the mean value; ordinate), and a cosine-fitting correlation score (correlation P value; abscissa) for each transcript under LL conditions. Each index is defined in the Materials and Methods. Briefly, a higher amplitude represents larger fluctuations, and a lower P value means a better correlation to a periodic (sinusoidal) waveform with a 24-h period. Seventy-eight “cycling genes” were extracted, with P values of <0.05 and amplitudes of >10−0.7 (within the red and yellow regions). For more stringent filtration, the lower amplitude and the higher P value from two independent experiments were used to ensure reproducibility. The circles indicate the all3173 and pecBAC (alr0523 to -0525) genes used for the bioluminescence reporter analysis and the kaiB gene. (B) Expression profiles of the 78 cycling genes sorted by peak time under LL conditions (left). The panel on the right shows a double plot of the expression profiles of the corresponding genes in one LD cycle reconstructed by connecting data from hour 4 to 12 in LL and hour 4 to 12 in the dark. The colors in descending order from red to black to green represent normalized data. The average and SD over two cycles are 0.0 and 1.0, respectively. The average levels in two independent experiments were used for the analysis. (C) Phase distributions of the peak expression times of 78 circadianly expressed genes. The numbers of ORFs from two independent experiments are plotted on the ordinate.
Fig 3
Fig 3
Temporal expression profiles of representative clock-controlled genes. The expression of photosynthesis-associated gap2 (all5062), encoding glyceraldehyde-3-phosphate dehydrogenase, and phycobilisome-related genes apcE (alr0020), apcC (asr0023), pecB (alr0523), cpcB (alr0528), and ho1 (all1897) peaked in subjective daytime, whereas rpl18 (all4200) and all4207 from a ribosomal gene cluster, secE (all5298), which is involved in protein secretion, and all1871, all3173, alr3692, groEL (alr3662), and all4578, encoding proteins with unknown functions, peaked in subjective night. For pecB and secE, we performed a Northern blot analysis with specific probes covering pecBAC (alr0523 to -0525) and all5298 and -5299, respectively. Note that pecB and all3173 were used for the bioluminescence reporter analysis. The pink and black lines represent the average expression levels calculated with Northern blot and microarray analyses, respectively. The dots represent the signals from two independent microarray experiments. The numbers on the ordinate indicate the relative expression levels based on the microarray analysis.
Fig 4
Fig 4
Circadian expression properties revealed with a bioluminescence reporter analysis. (A) Bioluminescence rhythms represent the clock-controlled promoter activities of the pecBACEF cluster (upper panel) and the all3173 gene (lower panel) from cells cultured on solid medium containing sodium nitrate in 35-mm culture plates under LL after two LD cycles. The bioluminescence profiles from four independent culture plates are shown (black traces). (B) Temperature compensation of the period. The bioluminescence profiles were monitored for the all3173 reporter strain, which was cultured on solid medium in the presence (solid line) or absence (dashed line) of sodium nitrate under LL after two LD cycles at 25, 30, or 35°C. The period length was plotted against temperature (n = 7 for each condition). The temperature coefficient Q10 values (the rate of change of a period length as a consequence of increasing the temperature by 10°C) in the presence and absence of combined nitrogen were 0.95 and 0.97, respectively. (C) Phase resetting of the all3173 bioluminescence rhythm in response to a 5-h dark pulse. At the circadian times indicated on the abscissa, the samples underwent 5 h of dark incubation, and they were then returned to LL to monitor the bioluminescence rhythm. The ordinate for each data point indicates the offset of the phase of the peaks after the treatment, relative to a control not pulsed with darkness, indicating a phase advance (positive values) or phase delay (negative values).
Fig 5
Fig 5
Nullification of transcriptional rhythms by genetic disruption of the kaiABC cluster. (A) Northern blot analysis using the total RNAs extracted from wild-type (WT) and kaiABC-null mutant (ΔkaiABC) cells with each kai gene-specific probe. (B) Bioluminescence rhythms to monitor the all3173 promoter activity, comparing the wild-type strain with the kaiABC-disrupted strain, in the presence of sodium nitrate. Results from a representative of multiple experiments are shown. (C) Circadian expression of the pecBAC, all3173, and groEL genes was nullified in the kaiABC-null mutant strains examined by Northern blot analysis. “Ctr” indicates a control RNA extracted from the wild-type strain at hour 64 in LL.
Fig 6
Fig 6
Circadian gene expression in heterocysts without combined nitrogen. (A) Variations in the changes in gene expression levels induced by nitrate deprivation and heterocyst enrichment. The abscissa shows the ratio of the average expression level for each gene throughout two circadian cycles under LL (hour 4 to 48) under nitrate deprivation (N−) to that in the presence of sodium nitrate (N+) (n = 2). The ordinate indicates the ratio of the average expression level for each gene 48 h after nitrate deprivation in heterocyst-enriched cells to that in intact bacterial filaments (n = 8). (B) Rhythmic expression of heterocyst-specific genes in LL under nitrate deprivation. Expression profiles of the all1427 and hesAB genes in the presence (black lines) or absence (pink lines) of sodium nitrate. Densitometric data from the Northern hybridization analysis (right panels) and microarray analysis are indicated by solid and dotted lines, respectively. For Northern blotting we loaded samples collected at hour 0 in the dark (D0) under different (N− for N+, N+ for N]) conditions as control samples, indicated by “Ctr.” (C) Expression of the all1427 and hesA genes in the wild-type strain (filled circles) and the hetR-null mutant strain (open circles) at 0, 3, 8, and 48 h after nitrate depletion. A densitometric analysis of the RT-PCR experiments is shown.

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