First selection: transformationPlating on plate with Km

Cultivationin liquid medium with Km

until the cells reach the stationary phase

Second selection:Plating on plate with Km

Colony PCR-1

Colony PCR-2

WT

ΔdnaA mutants

1 2 3 54 6 7 8

complete(18)

M2.691.881.490.93

kb WT Individual ΔdnaA mutants

Kmr

dnaA

1021 bp

1449 bp

incomplete(59)

b

DnaA

0 6 12 24 48 (h)

CBB

a

c

Total 77

Fig. S1

Segregation:

5'- CGCT T GCGGA A GA T CCGGA A A A T CA A GT T A C-3'

3'- GCGA A CGCCT T CT A GGCCT T T T A GT T CA A T G-5'

5'- AT CAA A A GCA GGA T CCA GA A A A T CCT A T T A T -3'

3'- T A GT T T T CGT CCT A GGT CT T T T A GGA T A A T A -5'

F RChr P PChr

c

F RChr P Chr P

Chr(0826)

Plasmid

b

oriC

ΔdnaA-1

aoriC

ΔdnaA-2

Fig. S2

oriC

0302

0680

1001

1294

1595

1943

2328

S. 7942genome

Per

cen

t re

cov

ery

α-FLAG

α-RpoD1

ΔdnaA

-1

WT

a b

c

Genomic position

ΔdnaA-1

ΔdnaA-2

Fig. S3

ΔdnaA-1

WT

ΔdnaA-2

AutofluorescenceDIC

SYTOXGreen merge

Fig. S4

Up: 27 geneDown: 91 gene

13

3

2

2

4

2

1

48

10

4

2

4

2

3

135

3

Fig. S5

Kmr

dnaA

1021 bp

1344 bp

gemome

WT

TK

Δd

na

AT

K

M

2.69

1.881.49

Kb

3.474.26

c

Specr

0964

792 bp

dnaN0963

Putative oriC region (POR)Containg DnaA box

M Kb

2.69

1.881.49

1

4

3

927 bp

d

e

2

3.474.26

M Kb

1.881.49

M Kb

1.881.49

Primer set

1, 2 1, 3 1, 4

WTTK

ΔdnaA

TK

ΔPORTK

CBB

α-HA

a b

f

Time (hr)

Ce

ll n

um

be

r (X

10

7 )W

TT

K

ΔP

OR

TK

WT

TK

ΔP

OR

TK

WT

TK

ΔP

OR

TK

ΔPORTK

WTTK

ΔdnaATK

Fig. S6

WT; 1280bpΔdnaA; 1379bp

genome

Nmr

dnaA dnaN

g h

pRL ２７１ WTTK

ΔdnaA

TK

1200

1517

1000900800

700

Blast search was performed to find the DnaA homologs in plants and algae using Synechococcus DnaA amino acid sequence data as the reference. Indicated data was top 10 in similarity. Upper, plants and algae; lower, cyanobacteria. DnaA protein is not conserved in all plants and photosynthetic prokaryotes, except for cucumber. All the homologous sequences of lower than 23 % indentity in plants and algae are ATPase domains, not DnaA protein-specific domains.

　 Species AnnotationQuery cover

E value Identity Accession

Plants and Algae

Cucumis sativusPREDICTED: chromosomal replication initiator protein DnaA-

like56% 1e-129 41% XP_004153787.1

Citrus sinensis hypothetical protein CISIN_1g0036231mg 41% 3e-23 23% KDO49346.1

Citrus sinensis hypothetical protein CISIN_1g0036231mg 41% 1e-22 23% KDO49342.1

Citrus sinensis hypothetical protein CISIN_1g0034991mg 36% 1e-22 23% KDO69546.1

Citrus sinensis hypothetical protein CISIN_1g0034991mg 36% 1e-22 23% KDO69542.1

Vitis vinifera PREDICTED: cell division cycle protein 48 homolog 47% 1e-22 22% XP_002276976.1

Citrus clementina hypothetical protein CICLE_v10018887mg 36% 1e-22 23% XP_006439879.1

Aegilops tauschii Cell division cycle 48-like protein 47% 1e-22 20% EMT25725.1

Citrus sinensis hypothetical protein CISIN_1g003620mg 41% 1e-22 23% KDO60203.1

Citrus sinensis PREDICTED: cell division cycle protein 48 homolog 47% 1e-22 23% XP_006476838.1

Cyanobacteria

Synechococcus elongatus PCC 6301 chromosomal replication initiator protein DnaA 100% 0.0 99% AP008231.1

Thermosynechococcus sp. NK55 chromosomal replication initiator protein DnaA 95% 0.0 73% CP006735.1

Geitlerinema sp. PCC 7407 chromosomal replication initiator protein DnaA 96% 0.0 73% CP003591.1

Crinalium epipsammum PCC 9333 chromosomal replication initiator protein DnaA 94% 0.0 74% CP003620.1

Thermosynechococcus elongatus BP-1

chromosomal replication initiator protein DnaA 96% 0.0 72% BA000039.2

Cyanothece sp. PCC 7425 chromosomal replication initiator protein DnaA 95% 0.0 73% CP001344.1

Synechococcus sp. PCC 6312 chromosomal replication initiator protein DnaA 96% 0.0 71% CP003558.1

Acaryochloris marina MBIC11017 chromosomal replication initiator protein DnaA 95% 0.0 71% CP000828.1

Oscillatoria nigro-viridis PCC 7112 chromosomal replication initiator protein DnaA 95% 0.0 69% CP003614.1

Gloeocapsa sp. PCC 7428 chromosomal replication initiator protein DnaA 95% 0.0 71% CP003646.1

Table S1

Table S2

Up regulated genesGene ID Gene name Product

Coenzyme transport and metabolismSynpcc7942_1188 Ap-4-A phosphorylase II-like proteinSynpcc7942_1282 moaA Molybdenum cofactor biosynthesis protein ASynpcc7942_1530 Molybdenum-pterin binding domain

Inorganic ion transport and metabolismSynpcc7942_1409 futA2 Iron transport system substrate-binding proteinSynpcc7942_2445 sphX Phosphate binding protein

Signal transduction mechanismsSynpcc7942_1148 Metal dependent phosphohydrolaseSynpcc7942_1525 GTP-binding protein TypA

Replication, recombination and repairSynpcc7942_0472 Exodeoxyribonuclease VII small subunitSynpcc7942_1842 Transposase, IS605 OrfB

transcriptionSynpcc7942_1159 Transcriptional regulator, MarR family

othersSynpcc7942_0900 gifA Hypothetical proteinSynpcc7942_1391 Mg chelatase-related proteinSynpcc7942_1969 Transcriptional regulator AbrBSynpcc7942_2306 Heat shock protein DnaJ-like

hypothetical proteinSynpcc7942_0013 Hypothetical proteinSynpcc7942_0014 Hypothetical proteinSynpcc7942_0457 Hypothetical proteinSynpcc7942_1150 Hypothetical proteinSynpcc7942_1161 Hypothetical proteinSynpcc7942_1209 Hypothetical proteinSynpcc7942_2088 Hypothetical proteinSynpcc7942_2167 Hypothetical proteinSynpcc7942_2336 Hypothetical proteinSynpcc7942_2392 Hypothetical proteinSynpcc7942_2483 Hypothetical proteinSynpcc7942_2586 Hypothetical protein

Table S2Down regulated genes

Gene ID Gene name Product Gene ID Gene name ProductPhotosynthesis Others

Synpcc7942_0325 apcC Lc 7.8 apoprotein (core components of the phycobilisomes) Synpcc7942_0426 Condensin subunit ScpB

Synpcc7942_0326 qpcB Allophycocyanin, beta subunit Synpcc7942_0855 PixG Response regulator receiver domain protein (CheY-like)

Synpcc7942_0327 apcA Allophycocyanin alpha chain Synpcc7942_1012 sphR Two component transcriptional regulator, winged helix family

Synpcc7942_0328 apcE Phycobilisome core-membrane linker polypeptide Synpcc7942_1205 Phage_integrase-like

Synpcc7942_1002 psaD Photosystem I reaction center subunit II Synpcc7942_1285 moaC Bifunctional molybdenum cofactor biosynthesis protein C/molybdopterin-binding protein

Synpcc7942_1050 Phycobilisome rod linker polypeptide Synpcc7942_1540 isiC Hypothetical protein

Synpcc7942_1051 cpcl2, cpcD Phycocyanin linker protein 9K Synpcc7942_1607 somA2 Probable porin; major outer membrane protein

Synpcc7942_1912 psaM Photosystem I reaction center subunit XII Synpcc7942_1649 Rubrerythrin

Synpcc7942_2010 psbV Cytochrome c550 Synpcc7942_2057 Acyl-phosphate glycerol-3-phosphate acyltransferase

Synpcc7942_1225 PcyA Phycocyanobilin:ferredoxin oxidoreductasehypothetical protein

ATP synthase Synpcc7942_0083 Hypothetical protein

Synpcc7942_0331 F0F1 ATP synthase subunit A Synpcc7942_0123 Hypothetical protein

Synpcc7942_0332 F0F1 ATP synthase subunit C Synpcc7942_0318 Hypothetical protein

Synpcc7942_0334 F0F1 ATP synthase subunit B Synpcc7942_0342 Hypothetical protein

Synpcc7942_0335 F0F1 ATP synthase subunit delta Synpcc7942_0414 Hypothetical proteinSynpcc7942_0415 Hypothetical protein

Amino acid transport and metabolism Synpcc7942_0551 Hypothetical protein

Synpcc7942_0934 cnaT Hypothetical protein Synpcc7942_0593 Hypothetical protein

Synpcc7942_1034 Transglutaminase-like Synpcc7942_0630 Hypothetical protein

Synpcc7942_1276 Extracellular solute-binding protein, family 3 Synpcc7942_0646 Hypothetical protein

Synpcc7942_2052 Probable oligopeptides ABC transporter permease protein Synpcc7942_0769 Hypothetical proteinSynpcc7942_0770 Hypothetical protein

Carbohydrate transport and metabolism Synpcc7942_0795 Hypothetical protein

Synpcc7942_0603 glgc Glucose-1-phosphate adenylyltransferase Synpcc7942_0810 Hypothetical protein

Synpcc7942_0888 Mannose-1-phosphate guanylyltransferase-like Synpcc7942_0837 Hypothetical protein

Synpcc7942_0889 Hypothetical protein Synpcc7942_0840 Hypothetical proteinSynpcc7942_0849 Hypothetical protein

Inorganic ion transport and metabolism Synpcc7942_0850 Hypothetical protein

Synpcc7942_1462 irpA Imelysin. Metallo peptidase. MEROPS family M75 Synpcc7942_0907 Hypothetical protein

Synpcc7942_1656 katG Catalase/peroxidase HPI Synpcc7942_1032 Hypothetical protein

Synpcc7942_1858 ho1 Heme oxygenase (decyclizing) Synpcc7942_1033 Hypothetical proteinSynpcc7942_1042 Hypothetical protein

Secondary metabolites biosynthesis, transport and catabolism Synpcc7942_1267 Hypothetical protein

Synpcc7942_0089 Carboxymethylenebutenolidase Synpcc7942_1288 Hypothetical protein

Synpcc7942_1408 mapA Membrane-associated protein Synpcc7942_1350 Hypothetical proteinSynpcc7942_1363 Hypothetical protein

Coenzyme transport and metabolism Synpcc7942_1508 Hypothetical protein

Synpcc7942_1805 HetI protein-like Synpcc7942_1568 Hypothetical protein

Synpcc7942_1072 cobO Cob(I)yrinic acid a,c-diamide adenosyltransferase Synpcc7942_1655 Hypothetical proteinSynpcc7942_1712 Hypothetical protein

Lipid transport and metabolism Synpcc7942_1764 Hypothetical protein

Synpcc7942_0310 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase Synpcc7942_1770 Hypothetical proteinSynpcc7942_1797 Hypothetical protein

Cell cycle control, mitosis and meiosis Synpcc7942_1822 Hypothetical protein

Synpcc7942_1204 Prevent-host-death protein Synpcc7942_1871 Hypothetical proteinSynpcc7942_1913 Hypothetical protein

Energy production and conversion Synpcc7942_1914 Hypothetical protein

Synpcc7942_1461 irpB Thiol oxidoreductase-like Synpcc7942_2138 Hypothetical proteinSynpcc7942_2161 Hypothetical protein

Signal transduction mechanisms Synpcc7942_2281 Hypothetical protein

Synpcc7942_1537 stbB Hypothetical protein Synpcc7942_2357 Hypothetical proteinSynpcc7942_2408 Hypothetical protein

Replication, recombination and repair Synpcc7942_2419 Hypothetical protein

Synpcc7942_1867 Deoxyribodipyrimidine photo-lyase family protein Synpcc7942_2504 Hypothetical protein

Cell wall/membrane biogenesis

Synpcc7942_1887 wbdC Probable mannosyltransferase

Translation

Synpcc7942_1073 rph Ribonuclease PH

General function prediction only

Synpcc7942_0179 Secretion chaperone CsaA

Synpcc7942_0838 Elongator protein 3/MiaB/NifB

Synpcc7942_0839 Nitrilase

Others

Synpcc7942_0426 Condensin subunit ScpB

Synpcc7942_0855 PixG Response regulator receiver domain protein (CheY-like)

Synpcc7942_1012 sphR Two component transcriptional regulator, winged helix family

Synpcc7942_1205 Phage_integrase-like

Synpcc7942_1285 moaC Bifunctional molybdenum cofactor biosynthesis protein C/molybdopterin-binding protein

Synpcc7942_1540 isiC Hypothetical protein

Synpcc7942_1607 somA2 Probable porin; major outer membrane protein

Synpcc7942_1649 Rubrerythrin

Synpcc7942_2057 Acyl-phosphate glycerol-3-phosphate acyltransferase

Table S3

　 　for use Primer name sequence (5' to 3')

deletion cassette of dnaA gene in synechococcus

syfdnaA-us-F AATAATGCCCACCGTCACGGTGTTG

syfdnaA-us-RGCTCACAATTCCACACAGCGATTGCCAAAGCAGACAGGAC

syfdnaA-Kmr-FCTTTGGCAATCGCTGTGTGGAATTGTGAGCGGATAACAAT

syfdnaA-Km-RGGGTGGCCGTGGCTTTTAGAAAAACTCATCGAGCATCAAA

syfdnaA-ds-FCGATGAGTTTTTCTAAAAGCCACGGCCACCCCCTTTGCTCT

syfdnaA-ds-R TCAGCAGCACAGTTTTGGGCGGCGG

comfirmdnaA-us-F2 CTGCCGAGCCCCAAGGTTCGTTTGAAGAAG

dnaA-ds-R2 GCACACTCATGGGGGTTCACAGCACTCCGT

Southern blotting (plasmid probe)

probe1-F CTGTCAGAAGTGGAAAGTCAGTGAAC

probe1-R TTGACCAAGACTGCCAAGTTGTC

probe2-F CTGTGCTGTCTGTCCGATTGTG

probe2-R ACGGGAATAGAGTCAGTGGGAAC

probe5-R GGTTGCCAGCTTCCGGC

probe5-R GAGCTGACTTAGGGAATGGCTC

Southern blotting (chromosome probe)

probe3-F CAAATTCCCTGCGCTGCAAAAG

probe3-R GGTGCCGCTCAGTAAATCGAC

probe4-F CCGCCATGATGCTGCCTATC

probe4-R GAGGATCAATTAGCCTGCGCTG

probe6-F CCGGGACTGGATCTGAACAATC

probe6-R GGTGTACGCAAGATGGCGG

probe7-F GTACAGCAGACTCCTCAGATCAGT

probe7-R GCGACAGGTCAGGAGTAAACTACT

clonig for pColdTF dnaA-F-BamHI GGGCGGATCCATGCGACGCGGCAAAAAGGT

dnaA-R-SalI GGGCGTCGACCTAACCGCGGCTAGCTACAA

Synechocystis dnaA deletion cassette

syndnaA-us-F GGGCAAAGTGACAGGATTGGC

syndnaA-us-RCTCACAATTCCACACAAAAATATACAAATATACTTAG

syndnaA-Km-F TATATTTTTGTGTGGAATTGTGAGCGGATA

syndnaA-Km-R CCAGGGGTTTAGAAAAACTCATCGAG

syndnaA-ds-F TTTTTCTAAACCCCTGGCGATCG

syndnaA-ds-R ACCGAAGGCACCGTGGAG

Synechocystis 0964 deletion cassette

slr0964-us-F CAGTGTTAACGCCCGTCTGC

slr0964-us-R CTTCCCTCATCTTGATTAAGAGGCTAGGA

Spec-F TTAATCAAGATGAGGGAAGCGGTGATC

Spec-R CCCCTACTTATTTGCCGACTACCTT

slr0964-ds-F AAATAAGTAGGGGCCAGCGTTGC

slr0964-ds-R CCGCTTCCCCTTCATCGTTTTC

for confirmSlr0964-F CTGGCAGGGGTATTGCAGG

Slr0964-R GGGACAAAACAGAGATAACCCAGG

clonig for pTCTHTK-F-NdeI GGGCCATATGTTACGTTGACACCATCGAATG

TK-R-BglII GCCCAGATCTTCAGTTAGCCTCCCCCATCTC

Anabaena dnaA deletion

anadnaA-5F AAGAGCTCACCTGTGGCGCTGATAATTC

anadnaA-5R AAGGATCCACCACCTTTGCTGCTAATCG

anadnaA-3F AAGGATCCTGAGGAGTTTGGCGGTAAAG

anadnaA-3R AACTCGAGTAAACCTTCTCCGGTTGCTG

anadnaA-F TGTGACGATTAGCAGCAAAGG

anadnaA-R TCTTTACCGCCAAACTCCTCA

pATK constructionTK-F GTTAGCGCGAATTGATCTGG

TK-R TACGGCGTTTCACTTCTGAG