Schematic diagram for the sampling of cells under aerobic and microoxic conditions. (B) Gene set enrichment analysis on time-course transcriptome data. KEGG pathways enriched in upregulated or downregulated genes after 1-, 2-, and 4-h incubation under microoxic conditions are shown. (C) (left) Schematic diagram showing the classification of genes according to the time-course transcriptome. Transient (striped square), Plateau (open square), Continuous (filled square), and Late (dotty square) are denoted as all upregulated genes. (right) Heatmap showing expression change in all upregulated genes over the time-course. Genes were clustered into subgroups and sorted by the gene name. (D) The classified genes were mapped to the central carbon metabolism, centered on pyruvate. PEP: phosphoenolpyruvate, PYR: pyruvate, AcCoA: acetyl CoA, Ac-P: acetyl phosphate, OXA: oxaloacetate, PHB: polyhydroxy butyrate. rTCA: reverse tricarboxylic acid cycle.

List of transiently upregulated genes

The list of transiently upregulated genes was merged by transcriptional units and sorted by function. The transcriptional unit information was obtained from a previous study (Kopf et al., 2014).

(A) (left) Primary component scatter plot showing the profiles of RNA-seq data. (right) MA plot showing fold change (y-axis) and average (x-axis) of gene expression between wildtype and mutant strains at each timepoint. Red dots indicate defined DEGs (|Log2 FC| >1 with FDR < 0.05). (B) Log2 scaled expression fold change in genes in the hox and nifJ operons upon Δcyabrb2 and ΔsigE under aerobic conditions (0, 1 hour after microoxic condition (1 hr), and 2 hours after microoxic condition (2 hr). DEGs are marked with sky blue (downregulated upon deletion) or red (upregulated upon deletion). (C and D) Fraction of upregulated and downregulated genes upon the (C) Δcyabrb2 and (D) ΔsigE at the timepoints of aerobic conditions (0 hr), 1 hour after anoxic condition (1 hr), and 2 hour after anoxic condition (2 hr). Genes are classified according to Figure 1C. Asterisk (*) and dagger (†) denote statistically significant enrichment and anti-enrichment compared with all upregulated genes tested by multiple comparisons of Fisher’s exact test (FDR < 0.05).

Snapshot of ChIP-seq data for cyAbrB2 and cyAbrB1 under aerobic conditions. The heatmap in the second column indicates expression fold change upon Δcyabrb2 under aerobic conditions. Positive values (colored in red) indicate that the gene expression is higher in wildtype than in Δcyabrb2, and negative values (colored in blue) indicate the opposite. The positions for the insertion elements are marked with red in the third column. The heatmap in the fourth column indicates GC contents. High GC content are colored in blue and low GC contents are colored in blue.

(A) GC contents and region length of cyAbrB2 binding regions (black dots). The horizontal dotted line indicates the genomic average of GC content.

(B) Scatter plot of GC content and binding signal of cyAbrB2. The x-axis is the binding signal of cyAbrB2 in each 100 bp region, and the y-axis indicates GC contents within 500 bp windows sliding every 100 base pairs. CyAbrB2 binding regions are marked with red dots.

(C) Histogram of genes showing the extent of occupancy (not bound, partially overlapped, or entirely overlapped) by the cyAbrB2 binding region. The gray bars indicate non-IS genes, and the count numbers of the non-IS genes are displayed on the gray bars. The black bars indicate the IS genes, and the count numbers of the IS genes are displayed above the black bars.

(D) Boxplot showing fold change in gene expression by Δcyabrb2 under aerobic conditions. Genes are classified according to the extent of occupancy by the cyAbrB2 binding region. Asterisk (*) denotes statistical significance tested by multiple comparisons of the Wilcoxon-rank test.

(E) Fraction of genes overlapped or non-overlapped with cyAbrB2 binding regions at the timepoints of aerobic conditions. Genes are classified according to Figure 1C. Asterisk (*) denotes statistically significant enrichment compared with all upregulated genes tested by multiple comparisons of Fisher’s exact test.

(F) Distribution of cyAbrB2 around transiently upregulated genes. ChIP-seq data in aerobic (L + O2) and dark microoxic (D − O2) conditions are overlayed. Arrows with bold lines indicate transiently upregulated genes. Shaded arrows indicate operons whose data were obtained from a previous study. The bars below the graph indicate the binding regions of each protein. The black bar at the top of the figure indicates a length of 10 kbp.

(A) Venn diagram showing the number of peaks of SigE (left) and SigA (right) in aerobic (L + O2) and dark microoxic (D − O2) conditions. (B) Scatter plot showing changes in the binding signal of SigE and SigA by 1 hour cultivation under microoxic conditions. The binding signal of each 100 bp window is plotted. The binding signals are plotted in Figure 3E. (C) 2x2 tables showing co-occurrence of cyAbrB2 bindingregions and SigE peaks (top) or SigA binding peaks (bottom). Odds and p-values were calculated by Fisher’s exact test. (D) Distribution of ChIP enrichment signals of SigE and SigA peaks. The peaks were classified into two based on whether they overlapped with the cyAbrB2-binding region. (E) Snapshots of ChIP-seq data for CyAabrB2, SigE, and SigA at the nifJ region (top) and hox region (bottom). ChIP-seq data for cyAbrB2, SigE, and SigA under aerobic and dark microoxic conditions are overlayed. ChIP-seq data of cyAbrB2 under aerobic and microoxic conditions are colored blue and pink, respectively. ChIP-seq data for SigE and SigA are shown in solid lines (aerobic conditions) and filled lines (microoxic conditions). The positions of TSSs were obtained from a previous study (Kopf et al., 2014) and indicated by vertical dotted lines. Open triangles indicate peak summits under aerobic conditions, and solid triangles indicate peak summits under microoxic conditions.

(A) Schematic diagram of 3C analysis around hox operon. The black arrow shows the localiton of the bait primer, and white arrows ((a) to (n)) indicate loci where the interaction frequency with bait were assayed. Black arrowheads indicate the position of HindIII sites. ChIP-seq data of cyAbrB2 in the aerobic condition is displayed in the bottom, and cyAbrB2 binding regions are marked with shade. (B) and (C) The line plot showing the interaction frequency of each locus with hox fragment in the wildtype (B) and Δcyabrb2 (C). The solid, dashed, and dotty lines indicate data in the aerobic condition, 1hr, and 4hr cultivation of microoxic conditions, respectively. The line plots indicate the average interaction frequency over the random ligation (n=3). Indivisual data are plotted as dots. (D) Schematic diagram of the dynamics of transcription factors governing fermentative gene expression. (E) The model showing cyAbrB2 shapes higher order DNA structure of the cyanobacterial chromosome.

RT-qPCR validated the transiently upregulated genes classified by RNAseq. Transcripts extracted from wildtype (solid line), ΔsigE mutant (dotty line), Δcyabrb2 mutant (dashed line), and ΔsigE Δcyabrb2 double mutant (dot-dashed line) were assayed in the aerobic condition (0hr) and 1,2,4 hour incubation of microoxic conditions. As the representative of the transiently upregulated genes, expression of hoxF, hoxY, nifJ, and sll0744 were quantified by RT-qPCR. The line represents the mean of n=3, and individual data points are shown as dot plots. Data of each gene is normalized by the mean score of wildtypes in the aerobic condition.

Genotyping and the procedure for ChIP-seq of flag-tagged proteins

Validation of procedure for ChIP-seq of flag-tagged cyAbrB2, SigE, and SigA. (A) Confirmation of genomic deletion and the epitope tagging of abrB2 (#1-#3), the epitope tagging of abrB1 (#4 and #5), and deletion of sigE (#6 and #7). Dotty lines are homologous regions between plasmids and the genome. Arrows indicate position of check primers. (B) The immunoblot for inputs and immunoprecipitants of ChIP for cyAbrB2-FLAG and cyAbrB1-FLAG. Inputs equivalent to the indicated portion of IP were loaded. (C)Scatter plots showing the reproducibility of two replicates for ChIP-seq assay. ChIP-seq data of SigE, SigA, and cyAbrB2 in aerobic and microoxic conditions and ChIP-seq data of cyAbrB1 in the aerobic condition are shown. Dots indicate normalized IP read count / normalized input read count in each 100bp window. X-axis is the value of replicate1, and Y-axis is the value of replicate 2. (D) Reproducibility of ChIP-seq data of SigA and SigE, compared with the previous study (Kariyazono and Osanai 2022). (top) Pie charts show the overlapping of peaks called in this study and the previous study. (bottom) Scatter plot comparing ChIP binding signals of SigA and SigE peaks commonly called in present and previous studies. Plots boxed by dashed lines are peaks called only in the present or previous study.

Overview of ChIP-seq data

Overview for ChIp-seq of flag tagged cyAbrB2, cyAbrB1, SigE, and SigA. Y-axis indicates [normalized IP read count / normalized input read count at each 25bp window], and X-axis indicates chromosome position. (A) Distribution of cyAbrB2, cyAbrB1, SigE, and SigA across the whole genome of Synechocystis. Aerobic (L +O2) and dark microoxic (D -O2) data are displayed. (B) Magnified image for chromosome position of 1,550kb-1,800kb. ChIP-seq data of cyAbrB2, cyAbrB1, SigE, and SigA in aerobic and dark microoxic conditions are overlayed. The dots below the graph indicate the binding region of each protein calculated by peak caller.

GC content vs ChIP enrichment score of SigA and SigE

(A) Scatter plot showing GC contents in each 100bp vs. binding signal of SigA, SigE, and control IP. Data are displayed as in Figure 3C. (B) GC content in each 100bp of (left) SigE peaks and non-SigE peaks, and (right) SigA peaks and non-SigA peaks. (C) Venn diagram showing overlap of the binding region of cyAbrB1 and cyAbrB2 (left), and scatter plot showing ChIP binding signal of cyAbrB2 (y-axis) and cyAbrB1(x-axis) in the aerobic condition. Data is plotted as in Figure 4E.

Alteration of cyAbrB2 binding to genome under the microoxic condition

(A) Scatter plot showing changes of the binding signal by 1hr cultivation in the microoxic condition. The binding signal of each 100bp window is plotted. Red dots are cyAbrB2 binding regions in either aerobic or microoxic conditions. The dotty lines indicate Log2 fold enrichment of 0.5, 0, -0.5 between aerobic and microoxic conditions. (B) Amount of precipitated DNA by cyAbrB2 ChIP. Three experiments were performed in the aerobic and microoxic conditions. (C) Quantification for ChIP of cyAbrB2 by qPCR. The position of primers and ChIP-seq data of cyAbrB2 are shown at the top. (D) Western blot images of cyAbrB2-3FLAG. Proteins were extracted in the aerobic condition and 1hr and 4hr incubation under microoxic conditions. The total protein concentration of each sample was adjusted to 4mg/mL, measured by the BCA method. Quantification of cyAbrB2 from western blot image was performed by ImageJ (ver. 2.0.0-rc-65) and plotted in the right graph.

Validation of unidirectional primer sets for 3C assay

The validation of unidirectional primer sets for 3C assay is shown in Figure 5. The 3C sample in this assay is the mixture of all 3C samples assayed in Figure 5.

Dynamics of individual 3C scores

Re-plotting of Figure 5B and 5C with the x-axis showing time (0,1,4 hr in microoxic conditions) and the y-axis showing the interaction frequency. Plots from the individual samples are connected by solid (Wildtype) or dotty (Δcyabrb2) lines.

Fold changes of transcripts from sigA, sigB, sigC, sigD, and sigE

List of strains used in this study

plasmids used in this study

oligonucleotides used in this study