1. First selection: transformation Plating on plate with Km Cultivation in 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 12354 678 complete (18) M 2.69 1.88 1.49 0.93 kb WT Individual ΔdnaA mutants Km r dnaA 1021 bp 1449 bp incomplete (59) b DnaA 06122448(h) CBB a c Total 77 Fig. S1 Segregation:

2. 5'-CGCTTGCGGAAGATCCGGAAAATCAAGTTAC-3' 3'-GCGAACGCCTTCTAGGCCTTTTAGTTCAATG-5' 5'-ATCAAAAGCAGGATCCAGAAAATCCTATTAT-3' 3'-TAGTTTTCGTCCTAGGTCTTTTAGGATAATA-5' FR ChrP P c F R P P (0826) Plasmid b oriC ΔdnaA-1 a oriC ΔdnaA-2 Fig. S2

3. oriC 0302 0680 1001 1294 1595 1943 2328 S. 7942 genome Percent recovery α-FLAG α-RpoD1 ΔdnaA-1 WT ab c Genomic position ΔdnaA-1 ΔdnaA-2 Fig. S3

4. ΔdnaA-1 WT ΔdnaA-2 Auto fluorescence DIC SYTOX Green merge Fig. S4

5. Up: 27 gene Down: 91 gene 13 3 2 2 4 2 1 48 10 4 2 4 2 3 1 3 5 3 Fig. S5

6. Km r dnaA 1021 bp 1344 bp gemome WT TK ΔdnaA TK M 2.69 1.88 1.49 Kb 3.47 4.26 c Spec r 0964 792 bp dnaN0963 Putative oriC region (POR) Containg DnaA box M Kb 2.69 1.88 1.49 1 4 3 927 bp d e 2 3.47 4.26 M Kb 1.88 1.49 M Kb 1.88 1.49 Primer set 1, 21, 31, 4 WT TK ΔdnaA TK ΔPOR TK CBB α-HA a b f Time (hr) Cell number (X 10 7 ) WT TK ΔPOR TK WT TK ΔPOR TK WT TK ΔPOR TK WT TK ΔdnaA TK Fig. S6 WT; 1280bp ΔdnaA; 1379bp genome Nm r dnaA dnaN g h pRL ２７１ WT TK ΔdnaA TK 1200 1517 1000 900 800 700

7. 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. SpeciesAnnotation Query cover E valueIdentityAccession Plants and Algae Cucumis sativusPREDICTED: chromosomal replication initiator protein DnaA-like56%1e-12941%XP_004153787.1 Citrus sinensishypothetical protein CISIN_1g0036231mg41%3e-2323%KDO49346.1 Citrus sinensishypothetical protein CISIN_1g0036231mg41%1e-2223%KDO49342.1 Citrus sinensishypothetical protein CISIN_1g0034991mg36%1e-2223%KDO69546.1 Citrus sinensishypothetical protein CISIN_1g0034991mg36%1e-2223%KDO69542.1 Vitis viniferaPREDICTED: cell division cycle protein 48 homolog47%1e-2222%XP_002276976.1 Citrus clementinahypothetical protein CICLE_v10018887mg36%1e-2223%XP_006439879.1 Aegilops tauschiiCell division cycle 48-like protein47%1e-2220%EMT25725.1 Citrus sinensishypothetical protein CISIN_1g003620mg41%1e-2223%KDO60203.1 Citrus sinensisPREDICTED: cell division cycle protein 48 homolog47%1e-2223%XP_006476838.1 Cyanobacteria Synechococcus elongatus PCC 6301chromosomal replication initiator protein DnaA100%0.099%AP008231.1 Thermosynechococcus sp. NK55chromosomal replication initiator protein DnaA95%0.073%CP006735.1 Geitlerinema sp. PCC 7407chromosomal replication initiator protein DnaA96%0.073%CP003591.1 Crinalium epipsammum PCC 9333chromosomal replication initiator protein DnaA94%0.074%CP003620.1 Thermosynechococcus elongatus BP-1chromosomal replication initiator protein DnaA96%0.072%BA000039.2 Cyanothece sp. PCC 7425chromosomal replication initiator protein DnaA95%0.073%CP001344.1 Synechococcus sp. PCC 6312chromosomal replication initiator protein DnaA96%0.071%CP003558.1 Acaryochloris marina MBIC11017chromosomal replication initiator protein DnaA95%0.071%CP000828.1 Oscillatoria nigro-viridis PCC 7112chromosomal replication initiator protein DnaA95%0.069%CP003614.1 Gloeocapsa sp. PCC 7428chromosomal replication initiator protein DnaA95%0.071%CP003646.1 Table S1

8. Table S2 Up regulated genes Gene IDGene nameProduct Coenzyme transport and metabolism Synpcc7942_1188Ap-4-A phosphorylase II-like protein Synpcc7942_1282moaAMolybdenum cofactor biosynthesis protein A Synpcc7942_1530Molybdenum-pterin binding domain Inorganic ion transport and metabolism Synpcc7942_1409futA2Iron transport system substrate-binding protein Synpcc7942_2445sphXPhosphate binding protein Signal transduction mechanisms Synpcc7942_1148Metal dependent phosphohydrolase Synpcc7942_1525GTP-binding protein TypA Replication, recombination and repair Synpcc7942_0472Exodeoxyribonuclease VII small subunit Synpcc7942_1842Transposase, IS605 OrfB transcription Synpcc7942_1159Transcriptional regulator, MarR family others Synpcc7942_0900gifAHypothetical protein Synpcc7942_1391Mg chelatase-related protein Synpcc7942_1969Transcriptional regulator AbrB Synpcc7942_2306Heat shock protein DnaJ-like hypothetical protein Synpcc7942_0013Hypothetical protein Synpcc7942_0014Hypothetical protein Synpcc7942_0457Hypothetical protein Synpcc7942_1150Hypothetical protein Synpcc7942_1161Hypothetical protein Synpcc7942_1209Hypothetical protein Synpcc7942_2088Hypothetical protein Synpcc7942_2167Hypothetical protein Synpcc7942_2336Hypothetical protein Synpcc7942_2392Hypothetical protein Synpcc7942_2483Hypothetical protein Synpcc7942_2586Hypothetical protein

9. Table S2 Down regulated genes Gene IDGene nameProductGene IDGene nameProduct PhotosynthesisOthers Synpcc7942_0325apcCLc 7.8 apoprotein (core components of the phycobilisomes)Synpcc7942_0426Condensin subunit ScpB Synpcc7942_0326qpcBAllophycocyanin, beta subunitSynpcc7942_0855PixGResponse regulator receiver domain protein (CheY-like) Synpcc7942_0327apcAAllophycocyanin alpha chainSynpcc7942_1012sphRTwo component transcriptional regulator, winged helix family Synpcc7942_0328apcEPhycobilisome core-membrane linker polypeptideSynpcc7942_1205Phage_integrase-like Synpcc7942_1002psaDPhotosystem I reaction center subunit IISynpcc7942_1285moaCBifunctional molybdenum cofactor biosynthesis protein C/molybdopterin-binding protein Synpcc7942_1050Phycobilisome rod linker polypeptideSynpcc7942_1540isiCHypothetical protein Synpcc7942_1051cpcl2, cpcDPhycocyanin linker protein 9KSynpcc7942_1607somA2Probable porin; major outer membrane protein Synpcc7942_1912psaMPhotosystem I reaction center subunit XIISynpcc7942_1649Rubrerythrin Synpcc7942_2010psbVCytochrome c550Synpcc7942_2057Acyl-phosphate glycerol-3-phosphate acyltransferase Synpcc7942_1225PcyAPhycocyanobilin:ferredoxin oxidoreductase hypothetical protein ATP synthaseSynpcc7942_0083Hypothetical protein Synpcc7942_0331F0F1 ATP synthase subunit ASynpcc7942_0123Hypothetical protein Synpcc7942_0332F0F1 ATP synthase subunit CSynpcc7942_0318Hypothetical protein Synpcc7942_0334F0F1 ATP synthase subunit BSynpcc7942_0342Hypothetical protein Synpcc7942_0335F0F1 ATP synthase subunit deltaSynpcc7942_0414Hypothetical protein Synpcc7942_0415Hypothetical protein Amino acid transport and metabolismSynpcc7942_0551Hypothetical protein Synpcc7942_0934cnaTHypothetical proteinSynpcc7942_0593Hypothetical protein Synpcc7942_1034Transglutaminase-likeSynpcc7942_0630Hypothetical protein Synpcc7942_1276Extracellular solute-binding protein, family 3Synpcc7942_0646Hypothetical protein Synpcc7942_2052Probable oligopeptides ABC transporter permease proteinSynpcc7942_0769Hypothetical protein Synpcc7942_0770Hypothetical protein Carbohydrate transport and metabolismSynpcc7942_0795Hypothetical protein Synpcc7942_0603glgcGlucose-1-phosphate adenylyltransferaseSynpcc7942_0810Hypothetical protein Synpcc7942_0888Mannose-1-phosphate guanylyltransferase-likeSynpcc7942_0837Hypothetical protein Synpcc7942_0889Hypothetical proteinSynpcc7942_0840Hypothetical protein Synpcc7942_0849Hypothetical protein Inorganic ion transport and metabolismSynpcc7942_0850Hypothetical protein Synpcc7942_1462irpAImelysin. Metallo peptidase. MEROPS family M75Synpcc7942_0907Hypothetical protein Synpcc7942_1656katGCatalase/peroxidase HPISynpcc7942_1032Hypothetical protein Synpcc7942_1858ho1Heme oxygenase (decyclizing)Synpcc7942_1033Hypothetical protein Synpcc7942_1042Hypothetical protein Secondary metabolites biosynthesis, transport and catabolismSynpcc7942_1267Hypothetical protein Synpcc7942_0089CarboxymethylenebutenolidaseSynpcc7942_1288Hypothetical protein Synpcc7942_1408mapAMembrane-associated proteinSynpcc7942_1350Hypothetical protein Synpcc7942_1363Hypothetical protein Coenzyme transport and metabolismSynpcc7942_1508Hypothetical protein Synpcc7942_1805HetI protein-likeSynpcc7942_1568Hypothetical protein Synpcc7942_1072cobOCob(I)yrinic acid a,c-diamide adenosyltransferaseSynpcc7942_1655Hypothetical protein Synpcc7942_1712Hypothetical protein Lipid transport and metabolismSynpcc7942_1764Hypothetical protein Synpcc7942_03104-diphosphocytidyl-2-C-methyl-D-erythritol kinaseSynpcc7942_1770Hypothetical protein Synpcc7942_1797Hypothetical protein Cell cycle control, mitosis and meiosisSynpcc7942_1822Hypothetical protein Synpcc7942_1204Prevent-host-death proteinSynpcc7942_1871Hypothetical protein Synpcc7942_1913Hypothetical protein Energy production and conversionSynpcc7942_1914Hypothetical protein Synpcc7942_1461irpBThiol oxidoreductase-likeSynpcc7942_2138Hypothetical protein Synpcc7942_2161Hypothetical protein Signal transduction mechanismsSynpcc7942_2281Hypothetical protein Synpcc7942_1537stbBHypothetical proteinSynpcc7942_2357Hypothetical protein Synpcc7942_2408Hypothetical protein Replication, recombination and repairSynpcc7942_2419Hypothetical protein Synpcc7942_1867Deoxyribodipyrimidine photo-lyase family proteinSynpcc7942_2504Hypothetical protein Cell wall/membrane biogenesis Synpcc7942_1887wbdCProbable mannosyltransferase Translation Synpcc7942_1073rphRibonuclease PH General function prediction only Synpcc7942_0179Secretion chaperone CsaA Synpcc7942_0838Elongator protein 3/MiaB/NifB Synpcc7942_0839Nitrilase Others Synpcc7942_0426Condensin subunit ScpB Synpcc7942_0855PixGResponse regulator receiver domain protein (CheY-like) Synpcc7942_1012sphRTwo component transcriptional regulator, winged helix family Synpcc7942_1205Phage_integrase-like Synpcc7942_1285moaCBifunctional molybdenum cofactor biosynthesis protein C/molybdopterin-binding protein Synpcc7942_1540isiCHypothetical protein Synpcc7942_1607somA2Probable porin; major outer membrane protein Synpcc7942_1649Rubrerythrin Synpcc7942_2057Acyl-phosphate glycerol-3-phosphate acyltransferase

10. Table S3 for usePrimer namesequence (5' to 3') deletion cassette of dnaA gene in synechococcus syfdnaA-us-FAATAATGCCCACCGTCACGGTGTTG syfdnaA-us-R GCTCACAATTCCACACAGCGATTGCCAAAGCAG ACAGGAC syfdnaA-Kmr-F CTTTGGCAATCGCTGTGTGGAATTGTGAGCGGA TAACAAT syfdnaA-Km-R GGGTGGCCGTGGCTTTTAGAAAAACTCATCGAG CATCAAA syfdnaA-ds-F CGATGAGTTTTTCTAAAAGCCACGGCCACCCCC TTTGCTCT syfdnaA-ds-RTCAGCAGCACAGTTTTGGGCGGCGG comfirm dnaA-us-F2CTGCCGAGCCCCAAGGTTCGTTTGAAGAAG dnaA-ds-R2GCACACTCATGGGGGTTCACAGCACTCCGT Southern blotting (plasmid probe) probe1-FCTGTCAGAAGTGGAAAGTCAGTGAAC probe1-RTTGACCAAGACTGCCAAGTTGTC probe2-FCTGTGCTGTCTGTCCGATTGTG probe2-RACGGGAATAGAGTCAGTGGGAAC probe5-RGGTTGCCAGCTTCCGGC probe5-RGAGCTGACTTAGGGAATGGCTC Southern blotting (chromosome probe) probe3-FCAAATTCCCTGCGCTGCAAAAG probe3-RGGTGCCGCTCAGTAAATCGAC probe4-FCCGCCATGATGCTGCCTATC probe4-RGAGGATCAATTAGCCTGCGCTG probe6-FCCGGGACTGGATCTGAACAATC probe6-RGGTGTACGCAAGATGGCGG probe7-FGTACAGCAGACTCCTCAGATCAGT probe7-RGCGACAGGTCAGGAGTAAACTACT clonig for pColdTF dnaA-F-BamHIGGGCGGATCCATGCGACGCGGCAAAAAGGT dnaA-R-SalIGGGCGTCGACCTAACCGCGGCTAGCTACAA Synechocystis dnaA deletion cassette syndnaA-us-FGGGCAAAGTGACAGGATTGGC syndnaA-us-R CTCACAATTCCACACAAAAATATACAAATATACT TAG syndnaA-Km-FTATATTTTTGTGTGGAATTGTGAGCGGATA syndnaA-Km-RCCAGGGGTTTAGAAAAACTCATCGAG syndnaA-ds-FTTTTTCTAAACCCCTGGCGATCG syndnaA-ds-RACCGAAGGCACCGTGGAG Synechocystis 0964 deletion cassette slr0964-us-FCAGTGTTAACGCCCGTCTGC slr0964-us-RCTTCCCTCATCTTGATTAAGAGGCTAGGA Spec-FTTAATCAAGATGAGGGAAGCGGTGATC Spec-RCCCCTACTTATTTGCCGACTACCTT slr0964-ds-FAAATAAGTAGGGGCCAGCGTTGC slr0964-ds-RCCGCTTCCCCTTCATCGTTTTC for confirm Slr0964-FCTGGCAGGGGTATTGCAGG Slr0964-RGGGACAAAACAGAGATAACCCAGG clonig for pTCTH TK-F-NdeIGGGCCATATGTTACGTTGACACCATCGAATG TK-R-BglIIGCCCAGATCTTCAGTTAGCCTCCCCCATCTC Anabaena dnaA deletion anadnaA-5FAAGAGCTCACCTGTGGCGCTGATAATTC anadnaA-5RAAGGATCCACCACCTTTGCTGCTAATCG anadnaA-3FAAGGATCCTGAGGAGTTTGGCGGTAAAG anadnaA-3RAACTCGAGTAAACCTTCTCCGGTTGCTG anadnaA-FTGTGACGATTAGCAGCAAAGG anadnaA-RTCTTTACCGCCAAACTCCTCA pATK construction TK-FGTTAGCGCGAATTGATCTGG TK-RTACGGCGTTTCACTTCTGAG