1

Supplemental Materials and Methods

Protein, primers, strains and media

Wild-type and mutant DnaA proteins were purified from overproducing E.

coli cells using a method described previously (Nishida et al. 2002; Ishida et al. 2004;

Kawakami et al. 2005; Kawakami et al. 2006). DARS-candidate sequences were

amplified by PCR. FK7-4, FK7-7 and FK7-22 were amplified using Kohara phage λ

#201 (Kohara et al. 1987) and the following primers: SIS-1 and del-3 for FK7-4, SIS-6

and SIS-7 for FK7-7, SIS-6 and SIS-16 for FK7-22. FK7-21, FK7-8, FK7-9 and

FK7-13 were amplified using plasmids as template (pOA76 for FK7-21, pOA23 for

FK7-8, pOA24 for FK7-9, and pOA35 for FK7-13) and primers (SIS-6 and SIS-7).

Sequences of these primers and of primers used for derivatives of DARS1 and DARS2

are listed in Supplemental Table 3.

All bacterial strains used in this study are listed in Supplemental Table 4. M9

medium contained standard M9 medium salts (Miller, 1972) supplemented with 0.2%

glucose, 0.2% casamino acids, and 5 µg/ml thiamine.

Plasmids

pCL1920 was a gift from National Institute Genetics (Japan). pKD46 was a

gift from Dr. Wanner and Dr. Niki (Datsenko and Wanner 2000). FK7-7 DNA was

digested with BamHI and ligated into the BamHI and XhoI sites of pACYC177 after the

unique XhoI site was filled in using KOD polymerase (Toyobo Co.), resulting in

2

pOA21. A region within pOA21 was amplified using primers SIS-17 and SIS-18,

self-ligated after being filled in, and phosphorylated by T4 polynucleotide kinase,

resulting in pOA76. Regions within the Kohara phageλ#462 (Kohara et al., 1987) were

amplified using primers (Kaz-9 and MutH-2 for pOA54, MutH-4 and MutH-2 for

pOA61, MutH-5 and MutH-2 for pOA62, and Kaz-9 and MutH-6 for pOA63), and the

resultant DNA fragments were digested with HindIII and BamHI, and cloned into the

HindIII and BamHI sites of pACYC177, resulting in pOA54, pOA61, poA62 and

pOA63. pOA54 was digested with BmgB1 and BamHI, the BamHI terminus was filled

in, and the resultant DNA fragment was self-ligated, resulting in pOA64. The fragment

amplified using pOA61 and a primer pair of MutH-14 and MutH-15 was self-ligated

after being filled in, and phosphorylated, resulting in pOA71. Similarly, pOA81, pOA82

and pOA83 were constructed using pOA61 and primer pairs of MutH-32 and MutH-33,

MutH-34 and MutH-35, and MutH-36 and MutH-37, respectively. A 0.45-kb

HindIII–BamHI fragment bearing minimal DARS2 or DARS2 mutant lacking DnaA

box-cluster was isolated from pOA61 or pOA71, respectively, and inserted into the

corresponding sites of pBR322, resulting in pKX11 or pOA77, respectively. Fragments

carrying mutations in DARS1 were prepared by annealing complementary

oligonucleotides (fk-7 and fk-8 for DnaA box I mutation, fk-9 and fk-10 for DnaA box

II mutation, fk-17 and fk-18 for DnaA box III mutation). The resultant DNA fragments

were cloned into the XhoI and BamHI sites of pACYC177, resulting in pOA23, pOA24

and pOA35, respectively.

3

Construction of ΔDARS1::kan, ∆DARS2::cat, and ∆DARS2::spec mutants

The chromosomal region corresponding to FK7-4 (Fig. 1) was replaced with

the kanamycin-resistant (kan) gene. Briefly, a downstream region of DARS1 was PCR

amplified using MG1655-genomic DNA and primers (Del-6 and Del-7). The resultant

DNA fragment was digested with SalI and SphI, and cloned using the corresponding

sites on pBR322, resulting in pOA5. The kan gene was amplified by PCR using pUC4K

and primers (Del-4 and Del-5), digested with SphI and BamHI, and ligated into the

corresponding sites on pOA5, resulting in pOA6. An upstream region of DARS1 was

amplified similarly using primers (Del-1 and Del-2), and the resultant DNA fragment

was digested with NheI and BamHI and ligated into the corresponding sites on pOA6,

resulting in pOA7. pOA7 was digested with SalI and ScaI, and introduced into E. coli

strain ME9018 (recD::mini-tet). Colonies were formed on LB plates containing

kanamycin (50 µg/mL) and screened for ampicillin sensitivity. Replacement of DARS1

with kan was verified by PCR. The resultant mutation (∆DARS1::kan) was introduced

into MG1655, MK86, KW262-5 and MIT166 using P1 transduction, resulting in MIT17,

MIT47, MIT167 and MIT168, respectively. Deletion of DARS1 in the transductants

was verified by PCR.

A DARS2 region containing DnaA box I-III was replaced with the cat or spec

gene in strain MG1655 harboring pKD46 (λ Red expression plasmid), according to a

method described previously (Datsenko and Wanner 2000; Baba et al. 2006), resulting

in the ∆DARS2::cat or spec mutants. Briefly, DNA fragments were PCR amplified

using pACYC184 and primers MutH-9 and MutH-10 (for ∆DARS2::cat) or using

4

pCL1920 and primers MutH-12 and MutH-13 (for ∆DARS2::spec). The resultant DNA

fragments were introduced into MG1655 bearing pKD46. Each deletion was verified by

PCR. ∆DARS2::cat was introduced into MG1655, MIT17 and KW262-5 using P1

transduction, resulting in MIT78, MIT80 and MIT166, respectively. ∆DARS2::spec

was also introduced into MG1655, MIT17, MK86 and MIT47 using P1 transduction,

resulting in MIT84, MIT92, MIT86 and MIT88, respectively. Deletion of DARS2 in the

transcductants was verified by PCR. tnaA::Tn10-linked dnaA508 was introduced into

MG1655, MIT17, MIT78 and MIT80 using P1 transduction.

5

Supplemental Table 1 Initial screen for DnaA-ADP-releasing activities on E. coli chromosomal regions

Fragment Coordinate Length (bp) ADP-release (%) F.kaz-1 4205192 – 4206301 1110 0 F.kaz-2 3662320 – 3663336 1017 0

F.kaz-3 3598249 – 3599393 1145 0 F.kaz-4 3179168 – 3180202 1035 0 F.kaz-5 (DARS2) 2966905 – 2968201 1297 25

F.kaz-6 2301300 – 2302542 1243 0 F.kaz-7 (DARS1) 811854 – 812897 1044 95 F.kaz-8 2627269 – 2628437 1169 0

F.kaz-9 3923293 – 3924415 1123 0 F.kaz-10 3805647 – 3806911 1265 0 F.kaz-11 3918745 – 3919863 1119 0

F.kaz-12 3451739 – 3452799 1061 0 F.kaz-13 2172148 – 2173448 1301 0 F.kaz-14 3795465 – 3796469 1005 0

F.kaz-15 1708400 – 1709402 1003 0 F.kaz-16 1494955 – 1496335 1381 0 F.kaz-17 1170241 – 1171256 1016 0

F.kaz-18 526298 – 527655 1358 0 F.kaz-19 2301186 – 2302542 1357 0 F.kaz-20 616614 – 617705 1092 0

F.kaz-21 1210305 – 1211314 1010 0 F.kaz-22 1507067 – 1508187 1121 0 F.kaz-23 2488412 – 2489499 1088 0

F.kaz-24 3266298 – 3267419 1122 0 F.kaz-25 3456220 – 3457231 1012 0 F.kaz-26 3899984 – 3901151 1168 0

F.kaz-27 4503213 – 4504169 957 0 F.kaz-28 934706 – 935810 1105 0 F.kaz-29 3551302 – 3552382 1081 0

6

F.kaz-30 3749648 – 3750756 1109 0 F.kaz-31 4239845 – 4240868 1024 0 F.kaz-32 4254561- 4255576 1016 0

F.kaz-33 915771 – 916786 1016 9 F.kaz-34 1590065 – 1591132 1068 0 F.kaz-35 2267073 – 2268361 1289 0

F.kaz-36 2962883 – 2963910 1028 0 F.kaz-37 2968784 – 2969892 1109 0 F.kaz-38 3131874 – 3133057 1184 0

F.kaz-39 3329812 – 3330840 1029 0 F.kaz-40 3510043 – 3511087 1045 0 F.kaz-41 3558953 – 3560072 1120 0

F.kaz-42 3687202 – 3688279 1078 0 F.kaz-43 3787706 – 3788847 1142 0 F.kaz-44 635401 – 636415 1015 0

F.kaz-45 1124006 – 1125137 1132 0 F.kaz-46 1933193 – 1934269 1077 7 F.kaz-47 2336762 – 2337921 1160 0

F.kaz-48 3358459 – 3359473 1015 0 F.kaz-49 4390579 – 4391561 983 9 F.kaz-50 1475663 – 1476762 1100 0

F.kaz-51 4460262 – 4461270 1009 0 The indicated E. coli chromosomal regions were amplified by PCR, and the resultant DNA fragments (100 fmol) were incubated with [3H]ADP-DnaA (2 pmol) at 30°C for

15 min in buffer containing 2 mM ATP. DNA-dependent release of ADP is shown for each fragment (ADP release %). Error range is <10%.

7

Supplemental Table 2 Relative cell mass and DNA content in DARS mutant cells

Strain

oriC- dependent replication

DARS1

DARS2

Relative DNA per

cell

Relative mean cell

mass

Relative DNA per

mass MG1655 + + + [1.0] [1.0] [1.0] MIT17 + ∆ + 1.0 1.1 0.9 MIT78 + + ∆ 1.0 1.1 0.9 MIT80 + ∆ ∆ 1.0 1.5 0.7

KW262-5 - + + 1.3 1.2 1.1 MIT167 - ∆ + 1.4 1.2 1.2 MIT166 - + ∆ 1.3 1.1 1.2 MIT168 - ∆ ∆ 1.4 1.3 1.1

Cells of indicated strains were grown at 37°C in M9 medium. Cell size and DNA content were analyzed by flow cytometry. The values of obtained for strain MG1655

are defined as 1, and relative values are shown compared to this value. MIT17, MIT78, and MIT80 are derivatives of MG1655 (wild-type). MIT167, MIT166 and MIT168 are derivatives of KW262-5 (ΔoriC rnhA::Tn3).

8

Supplemental Table 3 List of oligonucleotides

Primer Sequence SIS-1 CCGCTCGAGCGTCTTCGATTGACTGCAA

Del-3 GGGGATCCGTGCAAGCCGCGTATTCTCTC SIS-6 GGGGATAGGGGCTGGAGACAG SIS-7 GGGGATCCGTGCAAGCCGCGTATT

SIS-16 CGGGATCCTCTAATACACATGGTTATCCA SIS-17 CTGTCTCCAGCCCCTATCCCC SIS-18 GAGTTAGAAAACACGAGGCAAGCG

MutH-2 CGGGATCCATTGCTTTTTAGGTTGCCG Kaz-9 GAGTGCTGACCAAATCGCCG MutH-6 CGGGATCCTAAACAGGATCTTACACCAT

MutH-4 CCCAAGCTTGAGGAAGGGGTGGATAGCC MutH-5 CCCAAGCTTCTACGGAATTACTACGGGA MutH-14 AAACGGCTATCCACCCCTTCC

MutH-15 CTACGGAATTACTACGGGAAAAC MutH-32 TAGTTAAACGGCTATCCACCCCTTCC MutH-33 TATCTTTTCTGTGGATAGAGTTGTGAAG

MutH-34 ATATCGAAAAGGTGAATAAAAACGGC MutH-35 AGTTAGTTGTGAAGAACTACGGAATTAC MutH-36 ATATCACTCTATCCACAGAAAAGGTG

MutH-37 AGTTCTACGGAATTACTACGGGAAAACCCG Del-6 CATGCATGCGCGAATTGGCGTTAAAGACGG Del-7 CGAGTCGACGTTTTCAATCCCCGAACAGTAGCC

Del-4 GGCGGATCCTTGTCGGGAAGATGCGTG Del-5 CGGGCATGCGGGAAAGCCACGTTGTGTC Del-1 CCTGCTAGCGCCTTACTGGCAATGCTTC

Del-2 CCCGGATCCTTGCAGTCAATCGAAGACG MutH-9 TCACAGTTATGTGCAGAGTTATAAACAGAGGAAGGG

GTGGATAGCCGTTTGAGAGCCTGAGCAAACTG

MutH-10 ATATCGGGCTTATTCAGAATGCTCCGGGTTTTCCCG

9

TAGTAATTCCGTAGACCTGTGACGGAAGATCAC MutH-12 TCACAGTTATGTGCAGAGTTATAAACAGAGGAAGGG

GTGGATAGCCGTTTACCTGATAGTTTGGCTGTG

MutH-13 ATATCGGGCTTATTCAGAATGCTCCGGGTTTTCCCGT AGTAATTCCGTAGGAAGCCAGGGCAGATCCG fk-7 TCGAGGGGATAGGGGCTGGAGACAGAACTATATCATTC

CTGTGGATAACCATGTGTATTAGAGTTAGAAAACACGAG GCAAGCGAGAGAATACGCGGCTTGCACG

fk-8 GATCCGTGCAAGCCGCGTATTCTCTCGCTTGCCTCGTGT

TTTCTAACTCTAATACACATGGTTATCCACAGGAATGATATAGTTCTGTCTCCAGCCCCTATCCCC

fk-9 TCGAGGGGATAGGGGCTGGAGACAGTTATCCACTATTC

CGATATAGTTCCATGTGTATTAGAGTTAGAAAACACGAGGCAAGCGAGAGAATACGCGGCTTGCACG

fk-10 GATCCGTGCAAGCCGCGTATTCTCTCGCTTGCCTCGTGT

TTTCTAACTCTAATACACATGGAACTATATCGGAATAGTGGATAACTGTCTCCAGCCCCTATCCCC

fk-17 TCGAGGGGATAGGGGCTGGAGACAGTTATCCACTATTC

CTGTGGATAACCAAACTATATCGAGTTAGAAAACACGAGGCAAGCGAGAGAATACGCGGCTTGCACG

fk-18 GATCCGTGCAAGCCGCGTATTCTCTCGCTTGCCTCGTGT

TTTCTAACTCGATATAGTTTGGTTATCCACAGGAATAGTG GATAACTGTCTCCAGCCCCTATCCCC

10

Supplemental Table 4 List of E. coli strains Strain Relevant genotype Source or reference DH5α F- endA1 hsdR17(rk

-mk+) supE44 thi1 Laboratory stock

recA1 gyrA(Nalr) relA1 ∆(lacZYA-argF) U169(φ80lacZ∆M15)

KA474 KA473a dnaN59a Kurokawa et al (1999) KH5402-1 thyA b Nishida et al (2002) YT411 KH5402-1 rnhA::cat Nishida et al (2002)

KA451 KH5402-1 rnhA::cat, dnaA::Tn10 Nishida et al (2002) KA429 KH5402-1 rnhA::cat, ∆oriC1071::Tn10 Nishida et al (2002) KA450 KH5402-1 ∆oriC1071::Tn10 Nishida et al (2002)

dnaA17 (Amber) rnhA199(Amber), WK001(pHCS4-1) KH5402-1 ∆hda::cat bearing pHCS4-1c (Fujimitsu et al. 2008) KP7364 KP245d ∆dnaA::spec rnhA::kan Miki, T.

(Kurokawa et al. 1999) ME9018 MG1655 recD1903::mini-tet NIGe MG1655 Wild type Laboratory stock

MIT17 MG1655 ∆DARS1::kan This work MIT78 MG1655 ∆DARS2::cat This work MIT84 MG1655 ∆DARS2::spec This work

MIT80 MIT17 ∆DARS2::cat This work MIT92 MIT17 ∆DARS2::spec This work KW262-5 MG1655 rnhA::Tn3 ∆oriC::tet (Kato and Katayama 2001)

MK86 KW262-5 ∆hda::cat (Kato and Katayama 2001) MIT47 MK86 ∆DARS1::kan This work MIT86 MK86 ∆DARS2::spec This work

MIT88 MIT47 ∆DARS2::spec This work MIT140 MG1655 dnaA508 tnaA::Tn10 This work MIT21 MIT17 dnaA508 tnaA::Tn10 This work

MIT141 MIT78 dnaA508 tnaA::Tn10 This work MIT142 MIT80 dnaA508 tnaA::Tn10 This work MIT167 KW262-5 ∆DARS1::kan This work

11

MIT166 KW262-5 ∆DARS2::cat This work MIT168 MIT166 ∆DARS1::kan This work a Other genetic markers are HfrC thyA metB uhp-1 rel-1 tnA22 (λ+). b Other genetic markers are ilv thr thrA(amber) trpE9829(amber) metE deo supF6(Ts). c pACYC177 derivatives carrying hda gene. d Other genetic markers are thyA trp his metB lac gal tsx. e National Institute of Genetics, Japan.

12

Supplemental References

Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., and Mori, H. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol

2: 2006 0008. Datsenko, K.A. and Wanner, B.L. 2000. One-step inactivation of chromosomal genes in

Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97(12):

6640-6645. Fujimitsu, K., Su'etsugu, M., Yamaguchi, Y., Mazda, K., Fu, N., Kawakami, H., and

Katayama, T. 2008. Modes of overinitiation, dnaA gene expression, and

inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia

coli. J Bacteriol 190(15): 5368-5381. Ishida, T., Akimitsu, N., Kashioka, T., Hatano, M., Kubota, T., Ogata, Y., Sekimizu, K.,

and Katayama, T. 2004. DiaA, a novel DnaA-binding protein, ensures the timely initiation of Escherichia coli chromosome replication. J Biol Chem 279(44): 45546-45555.

Kato, J. and Katayama, T. 2001. Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli. EMBO J 20(15): 4253-4262.

Kawakami, H., Keyamura, K., and Katayama, T. 2005. Formation of an

ATP-DnaA-specific initiation complex requires DnaA Arginine 285, a conserved motif in the AAA+ protein family. J Biol Chem 280(29): 27420-27430.

Kawakami, H., Ozaki, S., Suzuki, S., Nakamura, K., Senriuchi, T., Su'etsugu, M., Fujimitsu, K., and Katayama, T. 2006. The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1

motif. Mol Microbiol 62(5): 1310-1324. Kohara, Y., Akiyama, K., and Isono, K. 1987. The physical map of the whole E. coli

chromosome: application of a new strategy for rapid analysis and sorting of a

large genomic library. Cell 50(3): 495-508. Kurokawa, K., Nishida, S., Emoto, A., Sekimizu, K., and Katayama, T. 1999.

Replication cycle-coordinated change of the adenine nucleotide-bound forms of

DnaA protein in Escherichia coli. EMBO J 18(23): 6642-6652.

13

Miller J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Nishida, S., Fujimitsu, K., Sekimizu, K., Ohmura, T., Ueda, T., and Katayama, T. 2002.

A nucleotide switch in the Escherichia coli DnaA protein initiates chromosomal replication: evidnece from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo. J Biol Chem 277(17): 14986-14995.

14

Legends of Supplemental Figures

Supplemental Figure 1 Heat-liable activity of DARS2-stimulating crude extract.

A crude protein extract (2 µg as protein) was incubated at 60°C for the indicated

amount of time, and further incubated at 30°C for 15 min in buffer containing

[3H]ADP-DnaA (2 pmol) and 5 fmol of pACYC177 (vector; ○) or pOA54 (●).

Supplemental Figure 2 DARS2 can reactivate ADP-DnaA resulting from RIDA

in vitro.

A, Release of DnaA-bound ADP that was produced by RIDA. DARS2-dependent

release of DnaA-bound ADP produced by RIDA was determined as described in Figure

3B, with exception that products were further incubated with the indicated amounts of

pOA61 or pACYC177 (Vector) in the presence of crude protein extract (80 µg/mL).

ADP-DnaA constituted 94% of ATP-/ADP-DnaA after the RIDA reaction.

B, DARS2-driven reactivation of RIDA-produced ADP-DnaA. The first stage was

performed as described in Figure 3B. In the second stage, the samples that had been

incubated at 30°C with (red) or without (blue) the DNA-clamp complexes in the first

stage were further incubated at 30°C for 15 min with the indicated amounts of pOA61

or pACYC177 (Vector) in the presence of crude protein extract (80 µg/mL). After the

first or second stage, replication activity of DnaA was assessed in a minichromosomal

replication system as described in Figure 3C. Incorporation of nucleotides was 2 pmol

in the absence of DnaA or minichromosome (data not shown).

15

Supplemental Figure 3 Growth of colonies of hda::cat DARS mutant cells.

hda::cat was transduced into MG1655 (wild-type), MIT17 (∆DARS1::kan), MIT84

(∆DARS2::spec), MIT92 (∆DARS1::kan ∆DARS2::spec) by P1 phage. These

transdutants were incubated at 37°C for 20 h on LB agar plates containing

chloramphenicol (20 µg/ml) and were photographed.

Supplemental Figure 4 Cellular ATP-DnaA levels in ∆DARSs mutant cells carrying

rnhA::Tn3 ∆oriC::Tn10 ∆hda::cat mutations.

A representative original data for Fig. 5B is shown. KW262-5, rnhA::Tn3 ∆oriC::Tn10.

MK86, rnhA::Tn3 ∆oriC::Tn10 ∆hda::cat. MIT47, rnhA::Tn3 ∆oriC::Tn10 ∆hda::cat

∆DARS1::kan. MIT86, rnhA::Tn3 ∆oriC:: Tn10 ∆hda::cat ∆DARS2::spec. MIT88,

rnhA::Tn3 ∆oriC::Tn10 ∆hda::cat ∆DARS1::kan ∆DARS2::spec. Pre, Pre-immune

serum for control. α-DnaA, anti-DnaA serum. ND, none detected.

Supplemental Figure 5 Immunoblot analysis using anti-DnaA anitiserum.

MG1655 (wild-type), MIT17 (∆DARS1::kan), MIT78 (∆DARS2::cat) and MIT80

(∆DARS1::kan, ∆DARS2::cat) strains were grown as described for Figure 6B. The

cellular amounts of DnaA were determined as in Figure 4E. Relative ratios of DnaA

content of the indicated cells to that of the wild-type strain are shown at each

temperature. Similar results were obtained from three independent experiments.

Supplemental Figure 6 A model for DARS mechanism in DnaA reactivation.

16

Possible cyclic reaction to reactivate DnaA (see text for details).

Supplemental Figure 7 Bacterial DnaA box-clusters similar to DARS of E. coli

DnaA box-clusters were identified in chromosomal sequences of 34 eubacterial species

using the Blast search tool and the NCBI database. Arrows indicate the direction of the

DnaA box. DnaA boxes are marked with rectangles. Sequences that differ from the

consensus DnaA box (TTATNCACA) are in lowercase. E. coli, Escherichia coli K-12;

S. flexneri, Shigella flexneri 2a 2457T; S. typhimurium, Salmonella typhimurium LT2;

P. luminescens, Photorhabdus luminescens TTO1; Y. peptis, Yersinia peptis KIM; E.

carotovora atroseptica, Erwinia carotovora atroseptica SCRI1043; S. denitrificans,

Shewanella denitrificans OS217; V. cholerae, Vibrio cholerae El Tor N16961; A.

hydrophila subsp, Aeromonas hydrophila subsp. Hydrophila ATCC 7966; H. influenzae,

Haemophilus influenzae 86 028NP; C. psychrerythraea, Colwellia psychrerythraea

34H; P. atlantica, Pseudoalteromonas atlantica T6c; X. oryzae, Xanthomonas oryzae

KACC10331; X. fastidiosa, Xylella fastidiosa 9a5c; P. syringae, Pseudomonas syringae

pv B728a; C. burnetii, Coxiella burnetii RSA 493; L. Pneumophila Lens, Legionella

Pneumophila Lens; N. eutropha, Nitrosomonas eutropha C91; N. meningitidis,

Neisseria meningitidis FAM18; J. sp. Marseille, Janthinobacterium sp. Marseille; B.

mallei, Burkholderia mallei ATCC 23344; B. bronchiseptica, Bordetella bronchiseptica

RB50; D. acidovorans, Delftia acidovorans SPH-1; S. sp. TM1040, Silicibacter sp.

TM1040; B. bacilliformis, Bartonella bacilliformis KC583; G. sulfurreducens,

Geobacter sulfurreducens PCA; M. tuberculosis, Mycobacterium tuberculosis

17

CDC1551; B. subtilis, Bacillus subtilis subsp. subtilis str. 168; S. aureus,

Staphylococcus aureus RF122; S. coelicolor, Streptomyces coelicolor A3(2); S. tropica,

Salinispora tropica CNB-440; S. pneumoniae, Streptococcus pneumoniae TIGR4; L.

brevis, Lactobacillus brevis ATCC 367; A. oremlandii, Alkaliphilus oremlandii OhILAs;

and H. aurantiacus, Herpetosiphon aurantiacus ATCC 23779.

Supplemental Figure 1Fujimitsu_Supplemental Fig. 1

3244

Boun

d AD

P (p

mol

)

00.10.20.30.40.50.6

0 5 10 15 20Time at 60 °C (min)

pACYC177

pOA54

DN

A re

plic

atio

n (p

mol

)

ATP

ATP

ATP

ADP

ATP

ATP

ATP

ADP

0

0 00.1

0.05

0.2

0.3

0.3

0.3

0.3

0.15

30

DNA-clampInput DnaA form

Incubation (°C)

Plasmid (nM)

pOA61 Vector

30

Rec

over

y of

AD

P-D

naA

(%)

Plasmid (nM)

A

B

1st stage

2nd stage(30 °C)

2nd stage1st stage

020406080

100

0 0.1 0.2 0.3

Vector

pOA61

0

50

100

150

200

300

Supplemental Figure 2Fujimitsu_Supplemental Fig. 2

Supplemental Figure 3 Fujimitsu_Supplemental Fig. 3

DARS1DARS2

MG1655++

MIT17∆+

MIT84+∆

MIT92∆∆

Supplemental Figure 4

+++

+++

∆++

∆∆+

∆+∆

∆∆∆

hdaDARS1DARS2

Pre α-DnaA

ND 25 71 57 41 30

ATP

ADP

Fujimitsu_Supplemental Fig. 4

ATP/(ATP+ADP) on DnaA (%)

DARS130°C 37°C 42°C DnaA (ng)

DARS2

Relative DnaA:

++

∆+

+∆

∆∆

++

∆+

+∆

∆∆

++

∆+

+∆

∆∆ 0 0.

40.

81.

53.

0

1.1

1.0

[1.0

]

[1.0

]

[1.0

]

1.1

1.0

0.9

1.0

1.0

0.9

0.9

Supplemental Figure 5 Fujimitsu_Supplemental Fig. 5

Heat-dependentconformational change

Dissociation of apo-DnaAand

ATP binding to apo-DnaAFormation of a specific complex

DB I

DB IIDB III

ADP

ADPADP

ADP

ADP

ADP

DB I

DB IIDB III

ADP

ADP

ATP

ATPADPADP

Release of DnaA-bound ADP

Dissociation?

Stabilization?

Supplemental Figure 6Fujimitsu_Supplemental Fig.6

Supplemental Figure 7

C. psychrerythraea TTAG TTATCCACgATTCCTGTGGATAACTATGTGGActA AAGTP. atlantica AAGG TTATCCACtATATCTGTGGATAACTATGTGCATcA TTCAX. oryzae AGGT TTATCCACACCGCCTGTGGATAAAGCTGTGAATtA GTCTX. fastidiosa ACCT TTATCCACAATAATTGTGGATAACCTTGTtAATAA GACAP. syringae AAGG TTATCCACgCATACTGTGGATAACCTTGTGAAcAg CGGCC. burnetii CCCC TTATTCACAATTTCTGTGGATAACTTTGTGAgTAA TTTG L. Pneumophila Lens CTAC TTATCCcCATTTTCTGTGGATAACTCTGTGAATAc ACTCN. eutropha AAGG TTATACACAATTCCTGTGGATAACTATGTGAATAA ATCCN. meningitidis CCGG TTtTAaAaAATAA-TGTGGATAACTTTGTGGATAA TTTTJ. sp. Marseille TTAA cTATCCACAGCTGCTGTGGATAACTTTGTGGATAA GGGCB. mallei CAAG TTATCCACAGTTTCTGTGGAaAACTGTGTGGAaAt CTGGB. bronchiseptica AAAA cTATCCACAGAAATTGTGGATAATTCTGTGCAaAA CTTGD. acidovorans GGGT aTATCgtCAATTTCTGTGGATAACTTTGTGGggAt TATTS. sp. TM1040 TGAT gaATCCACcATTCCTGTGGATAACAATGcGGgTAA CTTTB. bacilliformis ATTA aaATCCACAATTCTTGTGGATAACTATcTGGATtt CATAG. sulfurreducens CCAA TTtTCCACAAAAAATGTGGATAACC-TGTtGATAA CGAT

B. Other positions

C. Other positions

E. coli GTTT TTATTCACcTTTTCTGTGGATAgAGTTGTGAAgAA CTACS. flexneri GTTT TTATTCACcTTTTCTGTGGATAgAGTTGTGAAgAA CTACS. typhimurium CATT TTATTCACcTTTTCTGTGGATAtAGTTGTGAAgAA GTATP. luminescens GCTT TTATTCACtTTTTCTGTGGATAtCTATGTGAAaAA GGCAY. peptis TATT TTATTCACtTTTTCTGTGGATAtAGGTGTGCAgAA CTCAE. carotovora atroseptica CAGT TTATTCACtTTTTCTGTGGAcAtCTTTGTGCAgAA CCCAS. denitrificans GGGA TTATCCACATTTTCTGTGGATAtCTCTGTtCgaAA CTTGV. cholerae GCAT TTATTCACgTTTTCTGTGAATAACCGTGTGAAgAA ACTTA. hydrophila subsp GCAG TTATCCAagATTTCTGTGGATAACTGTGTGCAacA GCGC

B. Upstream mutH (DARS2 in E. coli)

A. Upstream uvrB (DARS1 in E. coli)

E. coli ACAG TTATCCACtATTCCTGTGGATAACCATGTGTATtA GAGTS. flexneri ACAG TTATCCACtATTCCTGTGGATAACCATGTGTATtA GAGTS. typhimurium GCAG TTATCCACtATTCCTGTGGATAACCTTGTGTATtA GAGTP. luminescens AGAG TTATCCACtATTCCTGTGGATAACCTTGTGTATtA GACTY. peptis TGAG TTATCCACtATTCCTGTGGATAACCTTGTGCATtA ACAAE. carotovora atroseptica GGAG TTATCCACcATTTCTGTGGATAACTATGTGCATtA TGGAS. denitrificans TGAG TTATCCACtATTTCTGTGGATAAACCTGTGTATtA CTCCV. cholerae TCAC TTATCCACcAAAATTGTGGATAAGTGTGTtAATgg ATCTA. hydrophila subsp CTAC TTAcCCACAGAATCTGTGGATAACCATGTtCATgA AACTH. influenza TTTT cTATCCACaGAGCCTGTGGATAACTTTGTGGATtA ACTT

A. Upstream of dnaA

(II) Other bacteria

(I) Proteobacteria

M. tuberculosis GGTA cTgTCCACA---TTTGTGGATAgCCATGTGGAcAg TTCAB. subtilis CTCT TTATCCACA--AAGTGTGGATAA-GTTGTGGATtg ATTTS. aureus AAAG TTATCCACc--GATTGTtAtTAA-TTTGTGGATAA TTAT

S. coelicolor TTTC TTtTCCcCA --GCGTGTGGAcAATGCCTGTGGATAA CTCCS. tropica CTCG cTATCCACA --CCATGTGGATAgAACATGTGTAcgA CCACB. subtilis ATAA TTAcTCACA -GATATGTGGATAA AT-TGTGAATtg TCTGS. aureus CAAG TTATACACt GAAAATTGgGCATAA ATGTcaGGAaAA TATCS. pneumoniae TTTA TTATCCACA --ACCTGTGGATAA CTTTGTGAATAA GAGAL. brevis AAAG TTATCCACA GGTATTGgGGATAA --CTGTGGATAA TAAAA. oremlandii ATAG TTATTCACA --GTTTGTGGATAA CTATGTGAATAA CTATH. aurantiacus TGTA TTATACcatAGCTGCTTGTGGATAA CTAcGTGAATAA GCTG

Fujimitsu_Supplemental Fig. 7