CyAbrB2 is a nucleoid-associated protein in Synechocystis controlling hydrogenase expression during fermentation
- School of Agriculture, Meiji University, Japan
Figures
Figure 1 with 1 supplement
Time-course analysis of the transcriptome of Synechocystis on entry to the microoxic conditions.
(A) 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 …
Figure 1—figure supplement 1
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.
Figure 2 with 2 supplements
The impacts of ∆sigE and ∆cyabrb2 on the time-course transcriptome.
(A) 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 differentially expressed genes …
Figure 2—figure supplement 1
RT-qPCR validated the transiently upregulated genes classified by RNA-seq.
Transcripts extracted from wildtype (solid line), ∆sigE mutant (dotty line), ∆cyabrb2 mutant (dashed line), and ∆sigE ∆cyabrb2 double mutant (dot-dashed line) were assayed in the aerobic condition …
Figure 2—figure supplement 2
Primary component scatter plot showing the profiles of RNA-seq data.
Figure 3 with 5 supplements
The long-tract distribution of cyAbrB2 on the Synechocystis genome and the repressive effect of cyAbrB2 on the gene expression.
(A) Snapshot of ChIP-seq data for cyAbrB2 and cyAbrB1 under aerobic conditions. The heatmap in the second column indicates expression fold change upon ∆cyabrb2 under aerobic conditions. Positive …
Figure 3—figure supplement 1
Overview of genome occupancy of cyAbrB2, cyAbrB1, SigE and SigA under the aerobic and microoxic conditions.
(A and B) 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 25 bp window], and x-axis indicates …
Figure 3—figure supplement 2
Validation of procedure for ChIP-seq of FLAG-tagged cyAbrB2, SigE, and SigA.
(A) The immunoblot for inputs and immunoprecipitants (IP) of ChIP for cyAbrB2-FLAG and cyAbrB1-FLAG. Input lysate of untagged control (GT) is also loaded. Inputs equivalent to the indicated portion …
Figure 3—figure supplement 3
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 the position of check primers.
Figure 3—figure supplement 4
Relationships between GC content and binding patterns for SigE and SigA.
GC content vs ChIP enrichment score of SigA and SigE. (A) Scatter plot showing GC contents in each 100 bp vs. binding signal of SigA, SigE, and control IP. Data are displayed as in Figure 3C. (B) GC …
Figure 3—figure supplement 5
cyAbrB2 and cyAbrB1 show similar binding pattern and overlapping gene regulation.
(A) 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. …
Figure 4
Transient up-regulated genes are enriched in cyAbrB2 binding regions.
(A) Fraction of genes overlapped or non-overlapped with cyAbrB2 binding regions at the timepoints of aerobic conditions. Genes are classified according to Figure 1—figure supplement 1. Asterisk (*) …
Figure 5 with 2 supplements
Changes of cyAbrB2 binding pattern on entry to the microoxic condition.
(A) Scatter plot showing changes of the binding signal by 1 hr cultivation in the microoxic condition. The binding signal of each 100 bp window is plotted. Red dots are cyAbrB2 binding regions in …
Figure 5—figure supplement 1
Alteration of cyAbrB2 binding to genome under the microoxic condition.
(A) Amount of precipitated DNA by cyAbrB2 ChIP. Three experiments were performed in the aerobic and microoxic conditions. (B) Western blot images of cyAbrB2-3FLAG. Proteins were extracted in the …
Figure 5—figure supplement 2
Alteration of cyAbrB1 binding to genome under the microoxic condition.
(A) Amount of precipitated DNA by cyAbrB1 ChIP. Three experiments were performed in the aerobic and microoxic conditions. (B) Western blot images of cyAbrB1-3FLAG. The experiment and data analysis …
Figure 6 with 2 supplements
Sigma factors are excluded from cyAbrB2 binding regions.
(A and B) Anti-co-occurrence of cyAbrB2 binding regions and sigma factors. Mosaic plots of cyAbrB2 binding regions and SigE peaks (A) or SigA binding peaks (B) are shown. Odds and p-values were …
Figure 6—figure supplement 1
Changes of SigE and SigA distribution on the entry to the microoxic condition.
(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 …
Figure 6—figure supplement 2
Reproducibility of ChIP-seq data of SigA and SigE, compared with the previous study (Kariyazono and Osanai, 2022).
(Top) Venn diagrams 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 …
Figure 7 with 2 supplements
3C analysis showed changes of DNA conformation around hox and nifJ operon on entry to microoxic condition and the impact of cyabrb2 deletion on DNA conformation.
(A and F) Schematic diagram of 3C analysis around hox operon (A) and nifJ operon (F). In the panels (A) and (F), the black horizontal arrow shows the location of the bait primer, and white …
Figure 7—figure supplement 1
Dynamics of individual 3C scores.
Re-plotting of Figure 7 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 …
Figure 7—figure supplement 2
The validation of unidirectional primer sets for 3C assay is shown in Figure 7.
The 3C sample in this assay is the mixture of all 3C samples assayed in Figure 7.
Figure 8
Schematic diagram of the dynamics of transcription factors governing fermentative gene expression.
Tables
Table 1
List of transiently upregulated genes.
| Operon | ||
|---|---|---|
| Oxidoreductase | ||
| sll0741 | nifJ/‘pyruvate-ferredoxin/flavodoxin oxidoreductase’ | TU3296 |
| sll0743 | Hypothetical protein | |
| sll0744 | Dihydroorotate dehydrogenase (fumarate) | |
| sll1221 | hoxF/‘bidirectional [NiFe] hydrogenase diaphorase subunit’ | TU1714 |
| sll1222 | Unknown protein | |
| sll1223 | hoxU/‘bidirectional [NiFe] hydrogenase diaphorase subunit’ | |
| sll1224 | hoxY/‘NAD-reducing hydrogenase small subunit’ | |
| sll1225 | Unknown protein | |
| sll1226 | hoxH/‘NAD-reducing hydrogenase large subunit’ | |
| slr1434 | pntB/‘H+-translocating NAD(P) transhydrogenase subunit beta’ | TU1089 |
| Transporter | ||
| sll1450 | nrtA/‘nitrate/nitrite transport system substrate binding protein’ | TU1023 |
| sll1451 | nrtB/‘nitrate/nitrite transport system permease protein’ | |
| sll1452 | nrtC/‘nitrate/nitrite transport system ATP binding protein’ | |
| sll1453 | nrtD/‘nitrate/nitrite transport system ATP binding protein’ | |
| Two-component system | ||
| slr1214 | Twitching motility two-component system response regulator PilG | TU905 |
| slr1215 | Unknown protein | TU907 |
| Glycosyl transferase | ||
| slr2116 | spsA/‘spore coat polysaccharide biosynthesis protein; SpsA’ | TU1673 |
| Protease | ||
| sll1009 | frpC/‘iron-regulated protein’ | TU491 |
| Insertion sequence (transposase) | ||
| slr1523 | Transposase | TU1659 |
| sll1985 | Transposase | TU1589 |
| sll7001 | Transposase | NA |
| sll7003 | Toxin FitB | TU7001 |
| ssl0172 | Transposase | TU3163 |
| Other | ||
| slr1260 | Hypothetical protein | TU1446 |
| slr0668 | Unknown protein | TU3532 |
| slr5127 | Unknown protein | TU5127 |
| sll0710 | Unknown protein | TU97 |
| sll1307 | Unknown protein | TU1224 |
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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).
Table 2
Fold changes of transcripts from sigA, sigB, sigC, sigD, and sigE.
| 0 hr vs 1 hr | 1 hr vs 4 hr | ||||
|---|---|---|---|---|---|
| Sigma factor | Locus | Log2FC | FDR | Log2FC | FDR |
| SigA | slr0653 | –0.873248 | 0.00766486 | –0.0013514 | 0.99797563 |
| SigB | sll0306 | 1.38098826 | 8.42E-06 | 0.77453605 | 0.04057775 |
| SigC | sll0184 | 2.97101055 | 1.75E-16 | 1.30743549 | 0.00067892 |
| SigD | sll2012 | 0.4701823 | 0.1498473 | –0.4522181 | 0.32402556 |
| SigE | sll1689 | –1.9111759 | 1.96E-11 | –1.1223298 | 0.00633142 |
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Data is extracted from Supplementary file 1d.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Synechocystis sp. PCC6803) | Wildtype | https://doi.org/10.1016/0076-6879(88)67088-1 | GT | |
| Strain, strain background (Synechocystis sp. PCC6803) | ∆sigE::KmR | https://doi.org/10.1074/jbc.M505043200 | G50 | |
| Strain, strain background (Synechocystis sp. PCC6803) | SigA-8His-KmR | https://doi.org/10.1111/tpj.15687 | KR93 | |
| Strain, strain background (Synechocystis sp. PCC6803) | SigA-3FLAG-KmR | https://doi.org/10.1111/tpj.15687 | KR94 | |
| Strain, strain background (Synechocystis sp. PCC6803) | ∆cyabrb2::KmR | In this study | KR340 | The genome of GT strain was manipulated by the transformation of the plasmid VK203 |
| Strain, strain background (Synechocystis sp. PCC6803) | cyAbrB(sll0359)–3xFLAG-KmR | In this study | KR338 | The genome of GT strain was manipulated by the transformation of the plasmid VK200 |
| Strain, strain background (Synechocystis sp. PCC6803) | cyAbrB2(sll0822)–3xFLAG-KmR | In this study | KR339 | The genome of GT strain was manipulated by the transformation of the plasmid VK201 |
| Strain, strain background (Synechocystis sp. PCC6803) | ∆cyabrB2::KmR ∆sigE::CmR | In this study | KR359 | The genome of G50 strain was manipulated by the transformation of the plasmid VK82 |
| Recombinant DNA reagent | sigE∆CmR | In this study | VK82 | Plasmid backbone:pTA2 (Toyobo), available upon request |
| Recombinant DNA reagent | AbrB1-3F-KmR | In this study | VK200 | Plasmid backbone:pTA2 (Toyobo), available upon request |
| Recombinant DNA reagent | AbrB2-3F-KmR | In this study | VK201 | Plasmid backbone:pTA2 (Toyobo), available upon request |
| Recombinant DNA reagent | cyabrB2∆KmR | In this study | VK203 | Plasmid backbone:pTA2 (Toyobo), available upon request |
| Antibody | Anti-FLAG | Sigma-aldrich | F1804 | RRID:AB_262044 For immunoprecipitation |
| Antibody | Anti-FLAG (alkaline phosphatase conjugated) | Sigma-aldrich | A9469 | RRID:AB_439699 For western blot (1:20,000) |
Additional files
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Supplementary file 1
Oligonucleotides used in this study and the summary of NGS analysis.
(a) Oligonucleotides used in this study. (b) Numbers and percentages of NGS reads passed the processes. (c–e) Log2FC, LogCPM, LR, p-value, and false discovery rate (FDR) calculated by edgeR lrt method. (c) Processed data from time-course transcriptome for GT strain. (d) Processed data from the comparison between GT and sigE∆ strain in each timepoints. (e) Processed data from the comparison between GT and cyabrb2∆ strain in each timepoints. (f) List of SigE binding summit in the aerobic condition from ChIP-seq data. (g) List of SigE binding summit in the microoxic condition from ChIP-seq data. (h) List of SigA binding summit in the aerobic condition from ChIP-seq data. (i) List of SigA binding summit in the microoxic condition from ChIP-seq data. (j) List of cyAbrB2 binding region in the aerobic condition from ChIP-seq data. (k) List of cyAbrB2 binding region in the microoxic condition from ChIP-seq data. (l) List of cyAbrB1 binding region in the aerobic condition from ChIP-seq data. (m) Raw result of gene set enrichment analysis of time-course transcriptome (vs the aerobic condition).
- https://cdn.elifesciences.org/articles/94245/elife-94245-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/94245/elife-94245-mdarchecklist1-v1.docx
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Source data 1
Uncropped images for Figure 3—figure supplement 2 and 3, Figure 5—figure supplement 1 and 2, and Figure 7—figure supplement 2.
- https://cdn.elifesciences.org/articles/94245/elife-94245-data1-v1.zip
