modular evolution of tngbss, a new family of ices ......! 1! modular evolution of tngbss, a new...

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Modular evolution of TnGBSs, a new family of ICEs associating IS transposition, plasmid

replication and conjugation for their spreading

Supplementary materials

Supplementary figure legends

Figure S1: Diversity of TnGBS1 related ICEs. The annotated genes of nine TnGBS1 related

ICEs were aligned and compared by using the Easyfig software (1). Genes are shown as

arrows. The genes encoding the DDE transposase are in red. The genes coding for proteins

involved in mobilization/conjugation, replication, and surface adhesion are respectively in

green, orange and purple. The genes coding for a protein similar to the ClpA ATPase are in

pink. Other genes conserved in the nine TnGBS1-like ICEs are in grey and genes specific for

one or few ICEs are in white.

Figure S2: Diversity of TnGBS2 related ICEs. The annotated genes of 22 TnGBS2 related

ICEs were aligned and compared by using the Easyfig software (1). Genes are shown as

arrows. The genes encoding the DDE transposase are colored in red. The genes coding for

proteins involved in mobilization/conjugation, replication, and surface adhesion are

respectively colored in green, orange and purple. Genes specific for one or few ICEs are in

white.

Figure S3. Alignment of the right and left inverted repeats of TnGBS ICEs. Inverted

repeat (IR) sequences were aligned by using CLUSTALW (2) and the Logo generated by using

the WebLogo web site (3). ICE names are according to Table 1. Conserved bases are indicated

in blue and non conserved ones in red.

2

Figure S4: Phylogeny of TnGBS1 related conjugative module. Maximum likelihood (ML)

using PhyML on the phylogeny.fr platform (4) was used to infer phylogenetic relationships.

ML bootstrap support was determined using 100 bootstrap replicates. A. relaxase, B. VirB4

and C. VirD4 proteins.

Figure S5: Phylogeny of TnGBS2 related conjugative modules. Maximum likelihood (ML)

using PhyML on the phylogeny.fr platform (4) was used to infer phylogenetic relationships.

ML bootstrap support was determined using 100 bootstrap replicates. A. relaxase, B. VirB4

and C. VirD4 proteins

Figure S6: Schematic representation of TnGBS1 and the integrative plasmid

pSU18::erm-miniTnGBS1. A. Circular form and B. Inserted form of the non replicative

plasmid pSU18::erm-miniTnGBS1. C. Schematic representation of the circular and D. Inserted

copy of TnGBS1. Genes, the pACYC184 origin of replication and inverted repeats (IRs) are

indicated by arrows. The DNA fragment in pSU18::erm-miniTnGBS1 originating from

TnGBS1.1 is colored in orange. Oligonucleotides used for detection and quantification of the

circular form by Q-PCR are indicated by small arrows. O-11, O-12 oligonucleotides were used

to quantify the circular form and O-13, O-14 to quantify gb0410.

Figure S7. Sequences of the insertion sites of TnGBS1 and pSU18::erm-miniTnGBS1.

gbs numbers correspond to the nomenclature of the orthologous genes in strains NEM316.

Numbers of identified insertions per site are indicated on the right for 16 BM110 TCs and 61

and 23 transformants of pSU18::erm-miniTnGBS1 in NEM316 and in BM110, respectively.

Blue letters represent the right extremity of the transposable element (IR-R); Underlined

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letters, the sequence duplicated after insertion (DR); Red letters, putative -35 and -10

sequences; Purple letters, putative extended -10 region.

Figure S8: Phylogeny of TnGBS1 and TnGBS2 related conjugative modules and their

association with replication and/or integration modules. A. TnGBS1-like ICEs and related

plasmids. B. TnGBS2-like ICEs and related plasmids and ICEs. Phylogenies of the

conjugative modules correspond to the VirB4 phylogeny of the figures S5B (A) and S6B (B).

The presence of integration modules, DDE transposase or tyrosine recombinase and

replication modules of the RepA_N, RepE (PriCT family) or Rep_3 families (5) are indicated

on the right.

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Table S1. Bacterial strains and plasmids used for cloning and conjugation assays. Strains and plasmids

Relevant genotype and resistance profile 1 or characteristics

Reference or source

E. coli DH5α Invitrogen XL2-‐Blue Stratagene

S. agalactiae NEM316 Sequenced strain, carries TnGBS1 and

TnGBS2 (6)

WC3/UK21 Blood, EOD (7) 1122 Bovine origin (8) 2584 Bovine origin (8) CZ183 Bovine origin (9) GMP201 Erm; NEM316-‐TnGBS2::erm (10) GMP205 Erm; A909RF-‐TnGBS1::erm This work GMP212 Erm; BM110-‐TnGBS1::erm This work GMP206 Erm; NEM316-‐TnGBS1 repE::pG1 This work GMP207 Erm; NEM316-‐TnGBS2 repA::pG1 This work GMP208 Erm, Tet; BM110-‐TnGBS1 repE::pG1 This work GMP209 Erm, NEM316-‐TnGBS1 repE+::pG1 This work GMP210 Erm, NEM316-‐TnGBS2 repA+::pG1 This work GMP211 Erm,TcR, BM110 TC TnGBS1 repE+::pG1 This work BM110 Tet; Isolated from neonate blood culture (11) BM110 RF BM110 spontaneous mutant for Rif and Fus This work A909RF A909 spontaneous mutant for Rif and Fus (10)

S. gallolyticus UCN34

Tet

(12)

S. pyogenes BM137

Rif, Fus

(13)

E. faecalis JH2-‐2

Rif, Fus derivative of strain JH2

(14)

B. subtilis 168

Sequenced strain

(15)

Plasmids pSU18 Cm, pACYC184 derivative (16) pS18erm Cm, Erm This work pG1 Erm, thermosensitive replication (17) pTCVerm Erm, Km; oriR pACYC184, oriRpAMβ1 (18) pJIM2246-‐

Ptet Cm, derivative of pJIM2246 (19), inducible Ptet promoter

M-‐F Lartigue unpublished

1Amp, Erm, Fus, Rif, Tet, are resistance to ampicillin, erythromycin, fusidic acid, rifampicin, and tetracycline, respectively.

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Table S2: Oligonucleotides used in this study Name Sequence 5' => 3' Application

O-‐1 AAACTGCAGGGCTCCCTTCTTCATGAGATT pSU-‐erm::MiniTnGBS1 construction O-‐2 CGCGGATCCGATTGAGCGAAAGAGGTAGGG pSU-‐erm::MiniTnGBS1 construction O-‐3 CGGAATTCCGGTAAACTCTATGTTCAGATACAATTG Cloning TnGBS1.1 oriT region O-‐4 CGGGATCCCGATGGTCCGATGTCAATTTC Cloning TnGBS1.1 oriT region O-‐5 CTGAAAGAATTCTAATGACCTACTTATTTGAG Cloning TnGBS2.1 oriT region O-‐6 ACACAGGATCCCTTATTAAGTATTTGACAGTC Cloning TnGBS2.1 oriT region O-‐7 GCTTAGTTCTAATGATCCTTATA Insertion upstream rpsJ O-‐8 GATGTCTACCAAACGTTTGTG Insertion upstream rpsJ O-‐9 CGTTTATGGACTAACTTATTTGT Insertion upstream gbs0411 O-‐10 GTTTTTCCCAATCCATCAACAA Insertion upstream gbs0411 O-‐11 ACTGTCAAGGGCTGAGCAAG Circular form of TnGBS1.1 and Q-‐PCR O-‐12 TTTCTCCGTTCTCCCCATAA Circular form of TnGBS1.1 and Q-‐PCR O-‐13 GGGAGCTGAGACGATGAGTC Q-‐PCR, quantification gbs0410 O-‐14 CCGACCAAGTGTCTGGAGAT Q-‐PCR, quantification gbs0410 O-‐17 GGAACATTATGAAGCCGATGA Q-‐PCR, quantification polA O-‐18 TCAAACTCAGCAACTCCCTTT Q-‐PCR, quantification polA O-‐19 CGATTTTCTCGTTCTCTTAAGCTAT Q-‐PCR, quantification circular form TnGBS2.1 O-‐20 CGTTCTCTTAGGCGACAAGG Q-‐PCR, quantification circular form TnGBS2.1 O-‐21 TGAGGATAGTGGTTCACGACA Q-‐PCR, quantification gbs1118 O-‐22 AATCCTGCTGGAGCGTATTG Q-‐PCR, quantification gbs1118 O-‐23 CCGGAATTCTAAGTCTATCTGATTTATACAGTTAC repA inactivation by pG1 insertion O-‐24 CGCGGATCCTTACTGTAAAAGATGACTGTAGTCT repA inactivation by pG1 insertion O-‐25 CGGAATTCTAGGTGCTATCAGAGCCAGCTTA repE inactivation by pG1 insertion O-‐26 CGCGGATCCCTATCAGAAACTCGTCCTGAGAG repE inactivation by pG1 insertion O-‐27 ATAAGAATGCGGCCGCGTTGAAAGTGGGTGCTTGACA Cloning of TnGBS2 repA gene in pJIM2246-‐Ptet O-‐28 TTTCCCCGCGGTTCATCTTGTAACATGTTATTCCT Cloning of TnGBS2 repA gene in pJIM2246-‐Ptet O-‐29 ATAAGAATGCGGCCGCGATGCTAGTATTTAAGAAAGAGA Cloning of TnGBS1 repE gene in pJIM2246-‐Ptet O-‐30 TTTCCCCGCGGTGTCTTCAGTTACCCCAGCT Cloning of TnGBS1 repE gene in pJIM2246-‐Ptet O-‐31 ATTATATCATTGATAGAGTTATTTG pJIM2246-‐Ptet forward sequencing primer O-‐32 GCAGTGAGCGCAACGCAAT pJIM2246-‐Ptet reverse sequencing primer O-‐33 CCGGAATTCTAAAACTGGCGAGAATGAAGGTAA pG1 insertion downstream TnGBS2 repA gene O-‐34 CGCGGATCCTTAACGCATAACGCCAATTTGATC pG1 insertion downstream TnGBS2 repA gene O-‐35 CCGGAATTCTAAAAGGCTATCAGAGAAGAATTGA pG1 insertion downstream TnGBS1 repE gene O-‐36 CGCGGATCCTTAATCATTGATTGATAATCGTTTTCT pG1 insertion downstream TnGBS1 repE gene

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Table S3: TnGBS related ICEs ICE name1 Species -‐ Strain GC% ICE GC% host 3insertion site lenght kb accession number

TnGBS1 -‐ like TnGBS1 a to c S. agalactiae NEM316*4 37.6% 35.6% gbs0411-‐gbs0741-‐gbs0968 47 NC_004368 2TnGBS1.2 S. agalactiae ATCC 13813* -‐ 34.9% galE -‐ AEQQ01000019 TnGBS1.3 S. agalactiae FSL S3-‐026* 38.5% 36.1% rpsU 46 AEXT01000007 TnGBS1.4 a to b S. agalactiae FSL S3-‐026 38.5% 36.1% rpoB, polC 50 AEXT01000002, 8 TnSgallo1.1 S. gallolyticus ATCC 43143 38% 37.5% tRNA-‐Cys 51 AP012053 TnSpneum1.1 S. pneumoniae GA17328 37.1% 39.7% 5NI 46 AGPH01000017 TnSspX1.1 S. sp. 2_1_36FAA* 40.8% 40.5% glmS 40 NZ_GG704942 TnSsporal1.1 S. sp. oral taxon 071 str. 73H25AP 36.9% 41.5% rpsO 46 NZ_AEEP01000006 2TnSsang1.1 S. sanguinis SK1058* -‐ 43.1% pyrG -‐ AFBF01000017 TnSang1.1 S. anginosius F0211 34.6% 37.6% murF 52 AECT01000063 TnScanis1.1 S. canis FSL Z3-‐227 -‐ 39.7% rpmH -‐ NZ_AIDX01000002.1 2TnSinf1.1 S. infantis SK970* -‐ 39.4% rpsL -‐ AFUT01000004-‐08 TnSinf1.2 S. infantis X 37.1% 39.0% rpoE 44 AFUQ01000001 TnSpast1.1 S. pasteurianus ATCC 43144 -‐ 37.4 rpsP -‐ NC_015600 TnSinter1.1 S. intermedius JTH08 34.4% 37.7% purR 47 NZ_AIDX01000002.1 TnSuri1.1 S. urinalis 2285-‐97 30% 34.1% rodA 44 AEUZ01000004 2TnSsob1.1 S. sobrinus TCI-‐54 -‐ 43.5% eno -‐ AGGH01000002 2TnSsob1.2 S. sobrinus TCI-‐89 -‐ 43.5% rpmV -‐ AGGN01000040 2TnSsob1.3 S. sobrinus TCI-‐118 -‐ 43.5% rpmV -‐ AGHN01000009 2TnSsob1.4 S. sobrinus TCI-‐342 -‐ 43.5% dltA -‐ AGGT01000092 2TnSsob1.5 S. sobrinus TCI-‐384 -‐ 43.5% rpmV -‐ AGHI01000004 2TnSsob1.6 S. sobrinus TCI-‐61 -‐ 43.5% NI -‐ AGGJ01000234 2TnLsal1.1 L. salivarius ATCC11741 -‐ 32.5% NI -‐ ACGT01000030

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ICE name1 Species -‐ Strain GC% ICE GC% host 3insertion site lenght kb accession number TnGBS2 -‐ like TnGBS2 S. agalactiae NEM316* 38.0% 35.6% xpt 34 NC_004368 2TnGBS2.2 S. agalactiae ATCC 13813* -‐ 34.9% glyQ -‐ AEQQ01000097-‐98 TnGBS2.3 S. agalactiae Wc3 38.1% -‐ fbsA 33 This study TnGBS2.4 S. agalactiae CZ183 38.3% -‐ guaC 29 This study TnGBS2.5 S. agalactiae 1122 34.4% -‐ rpmH 38 This study TnGBS2.6 S. agalactiae 2584 34.5% -‐ rplJ 45 This study TnGBS2.7 S. agalactiae FSL S3-‐026* 38% 36.1% guaC 34 AEXT01000007 TnSinf2.1 S. infantis SK970* 39.1% 39.4% rpmV 32 AFUT01000008 2TnSinfta2.1 S. infantarius ATCC BAA-‐102 -‐ 37.6% rpmH NZ_ABJK02000018 TnSgallo2.1 S. gallolyticus UCN34 36.7% 37.6% rpmB 41 NC_013798 TnSgallo2.2 S. gallolyticus ATCC BAA-‐2069* 38.8% 37.8% rpmH 36 NC_015215 TnSgallo2.3 S. gallolyticus ATCC BAA-‐2069* 39.2% 37.8% NI 30 NC_015215 TnSoral2.1 S. oralis ATCC 35037 39.3% 40.3% NI 29 NZ_ADMV01000022 TnSoral2.2 S. oralis SK255 39.6% 41.5% HMPREF9968_1062 29 AFNM01000055 TnSsporal2.1 S. sp. oral taxon 071 str. 73H25AP 39.1% 41.5% glyQ 31 NZ_AEEP01000011 TnSspX2.1 S. sp. 2_1_36FAA* 41.6% 40.5% rpsF -‐ NZ_GG704942 TnSmit2.1 S. mitis SK597 37.6% 40.2% pyrF 30 NZ_AEDV01000027 2TnSmit2.2 S. mitis SK1073 -‐ 40.6% pyrF -‐ AFQT01000042 TnSsang2.1 S. sanguinis ATCC 49296 39.3% 41.6% rpmB 32 NZ_AEPO01000010 TnSsang2.2 S. sanguinis SK1059 38.9% 42.9% rpsB 25 AFFM01000023 2TnSsang2.3 S. sanguinis SK49 -‐ 42.9% relA2 -‐ AFFO01000015-‐16 TnSsang2.4 S. sanguinis SK72 41.7% 42.3% pyrG 25 AEXV01000011 2TnSsang2.5 S. sanguinis ATCC29667 -‐ 42.9% rpsB -‐ AFQA01000023-‐24 TnSsang2.6 S. sanguinis SK340 39.0% 42.0% rpsB 25 AFQB01000011 TnSsang2.7 S. sanguinis SK1058* -‐ 43.1% pyrG -‐ AFBF01000017 TnSsui2.1 S. suis D12 40.8% 41.3% rplU 33 CP002644 TnScons2.1 S. constellatus SK1060 38.2% 38.1% rpmH 25 NZ_AFUP01000009

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ICE name1 Species -‐ Strain GC% ICE GC% host 3insertion site lenght kb accession number 2TnSict2.1 S. ictaluri 707-‐05* -‐ 39.2% ABC transporter -‐ NZ_AEUX02000006 2TnSict2.2 S. ictaluri 707-‐05* -‐ 39.2% rimP -‐ NZ_AEUX02000007 TnSang2.1 S. anginosus SK52 37.8% 39.1% NI 33 NZ_AFIM01000003 TnSzoo2.1 S. equi subsp. zooepidemicus ATCC

35246 39.2% 41.6% rpmB 32 CP002904

TnSsob2.1 S. sobrinus TCI-‐396 41.1% 43.0% leuA 27 AGHL01000006 2TnSsob2.2 S. sobrinus TCI-‐345 -‐ 43.3% cspR -‐ AGGU01000185 2TnSsob2.3 S. sobrinus TCI-‐366 -‐ 43.6% leuA -‐ AGHB01000008 2TnSsob2.4 S. sobrinus TCI-‐377 -‐ 43.4% NI -‐ AGHG01000295 6TnSdys2.1 S. dysgalactiae subsp. equisimilis

GGS_124 35.6% 39.6% SDEG_1257 ABC transporter 6 NC_012891.1

6TnSdys2.2 S. dysgalactiae subsp. equisimilis ATCC 12394

35.7% 39.5% SDE12394_06580 6 AFUL01000001

1 TnGBS names include the abbreviation of the species followed by 1 or 2 which indicates whether the ICE is related to TnGBS1 or TnGBS2, respectively and by an order number 2 The TnGBS sequence is incomplete and/or split in several contigs. 3 The gene name corresponds to the promoter upstream of which the ICE is inserted. 4 Strains containing more than one TnGBS-‐ICE are indicated by a * 5 NI: not identifiable 6These two elements correspond to simple transposons.

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Figure S1

TnSgal1.1

TnSinf1.2

TnSpneu1.1

TnSsporal1.1

TnSspX1.1

TnGBS1.3

TnGBS1

TnGBS1.4

TnSang1.1

500 bp

Iden2ty % 20 to 100

Figure S2

Iden-ty % 20 to 100

500 bp

TnGBS2

TnGBS2.7

TnSzoo2.1

TnGBS2.3

TnGBS2.4

TnSang2.1

TnSgallo2.1

TnSsui2.1

TnGBS2.5

TnGBS2.6

TnSgallo2.3

TnSgallo2.2

TnSoral2.2

TnSsporal2.1

TnSoral2.1

TnSsang2.1

TnSmit2.1

TnScons2.1

TnSsang2.6

TnSspX2.1

TnSsang2.4

IR‐R IR‐L GTATGCGGTAGAATTTTTTAGAAT----ATTCTAAAAACTCTTACCACTTAC GTATGCGGTAGAATTTTTTAGAAT----TTTCTAAAAACTCTTACCACTTAC GTATGCGGTAGAATTTTTTAGAAC----TTTCTAAAAACTCTTACCACTTAC GTATGCGGTAGATTTTTTTAGAAC----TTTCTAAAAACTCTTACCACTTAC GTATGCGGTAGAATTTTCTAGAAC----TTTCTAAAAACTCTTACCACTTAC GTATGTGGTAGAATTTTTTAGAAT----ATTCTAAAAACTCTTACCACTTAC GTATGTGGTAGAATTTTTTAGAAT----TTTCTAAAAACTCTTACCACTTAC GTATGTGGTAGAATTTTTTAGAAT----ATTCTAAAAACTCTTACCACTTAC GTATGTGGTAGAATTTTTTAGAAA----TTTCTAAAAACTCTTACCACTTAC GTATGTGGAAGAATTTTTTAGAAT----ATTCTAAAAACTCTTACCACTTAC GTATGTGGTAAAATTTTTTAGAAT----ATTCTAAAAACTCTTACCACTTAC GAATGTGGTAAACTTTTGTAGAAA----ATTCTACAAACTCTTACCACTTAG GAATGTGGTAAACTTTTGTAGAAA----ATTCTACAAACTCTTACCACTTAG

TnGBS1.4 TnSpneu1.1 TnSinf1.1 TnSinf1.2

TnSsporal1.1 TnGBS1

TnGBS1.2 TnGBS1.3

TnSspX1.1 TnSuri1.1

TnSgallo1.1 TnSinter1.1

TnSang1.1

Figure S3

consensus

GTTTGTGTCTAAATTTTTGGGAAT----TTTCCCAAAACTCTAGACACTTAG GTTTGTGTCTAAATTTTTGGGAAT----TTTCCCAAAACTCTAGACACTTAC GTTTGTGTCTAAATTTTTGGGAAT----TTTCCCAAAACTCTAGACACTTAG GTTTGTGTCTAAATTTTTGGGAAT----TTTCCCAAAACTCTAGACACTTAC GTTTGTGTCTAAATTTTTGGGAAA----TTTCCCAAAACTCTAGACACTTAC GTATGTGTCTAAATTTCTGGGAAA----TTTCCCAAAACTCTAGACACTTAC GTATGTGTCTAAATTTCTGGGAAA----TTTCCCAAAACTCTAGACACTTAC GTATGTGTCTGAATTTTTGGGAAT----TTTCCCGAAACTCTAGACACTTAC GTATGTGTCTGAATTTTTGGGAAT----TTTCCCGAAACTCTAGACACTTAC GTATGTGTCTGAATTTTCGGGAAA----TTTCCCGAAACTCTAGACACTTAC GTTTGTGTCTGAATTTTTGGGAAA----TTTCCCGAAATTCTAGACACTTAC GTTTGTGTCTAAATTTTTGGGAAA----TTTCCCGAAACTCTAGACACTTAC GTATGTGTCTTTTTTTCTGGGAAT----TTTCCCAAAACTCTAGACACTTAC GTATGTGTCTTTTTTTCTGGGAAT----TTTCCCAAAACTCTAGACACTTAC GTATGTGTCTTTTTTTCTGGGAAA----TTTCCCAAAACTCTAGACACTTAC GTATGTGTCTGAATTTTTGAGAAG----TTTCTCAAAACTCTAGACACTTAC GTATGTGTCTGAATTTTTGAGAAG----TTTCTCAAAACTCTAGACACTTAC GTATGTGTCTGAATTTTTGAGAAG----TTTCTCAAAACTCTAGACACTTAC GTATGTGTCTCAATTTTTGAGAAG----TTTCTCAAAACTCTAGACACTTAC GTATGTGTCTCAATTTTTGAGAAG----TTTCTCAAAACTCTAGACACTTAC GTTTGTGTCTATTTTTTTGAGAAT----ATTCTCAAAAATCTATACACTTAC GTTTGTGTCTATTTTTTTGAGAAT----ATTCTCAAAAATCTATACACTTAC GTTTGTGTCTATTTTTTTGAGAAT----ATTCTCAAAACTCTAGACACTTAC GTATGTGTCGCGATTTTTGAGAAA----TTTCTCAAAACTCTAGACACTTAC GTATGTGTCTTATTTTTTGAGAAT----TTTCTCAAAACTCTAGACACTTAC GTATGTGTCTCAATTTTTGTGAAG----TTTCACAAAACTCTAGACACTTAC GAATGTGTATATTTTTTTGAGAAA----ATTCTCAAAACTTTATACACTTAC GAATGTGTCTGTTTTTGGGAGAAA----ATTCTCCCAATTTGGGACACATAC

TnSoral2.1 TnSoral2.2

TnSsporal2.1 TnSsang2.1 TnSsui2.1 TnSict2.1 TnSict2.2

TnGBS2 TnGBS2.7 TnGBS2.3 TnGBS2.4

TnSang2.1 TnSgallo2.1 TnSgallo2.3 TnSgallo2.2 TnSsang2.2 TnSsang2.5 TnSsang2.6 TnSsang2.3 TnSsang2.4

TnGBS2.2 TnGBS2.5

TnSinfanta2.1 TnSmit2.1 TnSsob2.1 TnSzoo2.1 TnGBS2.6

TnScons2.1

consensus

A

B

C

Figure S4

A

B

C

Figure S5

Targeted promoter Insertion site pSU::MiniTnGBS1 TC Total NEM316 BM110 BM110

gbs0057 rpsJ ACATACAATAAAAAAATAATGCTTGCGTGTGCTGTTTTTTTTTGATATCATATACTGG 2 2 gbs0104 pyrF ACATACTTTTTGCATAAAAACTTTGTAAAACGTTTTAAATTATGGTAAACTAAACTGT 1 1 gbs0125 rpsL ACATACATTTTCAGTAAAAAACTAGCATTTTTTATAGAATTGTGCTAGAATTAACATG 1 1 gbs0154 tyrS ACATACTCAGATTTAATATAACTTGCATATCCTAAAGAGTTTTGATATAGTTGACATT 1 1 gbs0156 ribD ACATACTCAAGAGAATAGAACTTGATTTATTGGTAAAAATAAGTTATAATGTATGTAA 2 2 1 5 gbs0258 glyQ ACATACAGAATAAGATAAATAGTAGACATAAAAGTCATTTTTTGTTATACTGTCTCAG 2 1 3 gbs0301 gmk ACATACAAAATAATAGGAGACCTTGAAAAAAAGGTTTCTTTTTGTTAAACTAATGAGA 5 5 gbs0353 ACATACCAGAATCAGCAAACTGTTGGAAGACAGAGATATTTCTGGTATAATATCTCTA 1 1 gbs0411 ACATACAAAATAAAGAAGATGATTGACTAAAGCCTATAAAACAAGTAAAATAAGAGGAA 1 1 gbs0443 rnpA ACATACTAACATTAGAAAAGAATTGAGTCTTTTAGGTTATTTATGGTATAATAAAACCA 1 1 2 gbs0494 ACATACCGATGTAGAAAGTGACTTTACTTATTTTTAAAAAACTGGTATAGTTTTATTTG 2 2 gbs0553 pyrD ACATACCATTATCATTATTTTCTTGCAGCTCTATTAATCAAGTGATAAAATAGTAAAAA 1 1 gbs0615 ACATACCCATATATGATATCGCTTTCATTCTTATGTTTTTTGTGATAAACTAATTAAAG 1 1 gbs0767 ribD ACATACAAACACATTATTTTTATTGACAAATAAAAATGAGTTTAGTAAAATAACTGTCA 4 2 6 gbs0772 ACATACATAGACTTTTCACCATTTGAAAATAAGGGTAGAAAAAGGTATAATTAACAATT 1 1 gbs0809 ACATACAGTTTATTGAAAGCGCTTGACAAGATTAGTAAGAAATGATAAAATTAAGACGT 1 1 gbs0838 ACATACGTTAGAAATAAAACACTTGCTTTTTCCATTCTTTTTTTATATGCTATTACCAT 1 1 gbs0842 ACATACAGAAATTAATTATAGGTATTTTTAGTCTAATTATTATAGCGATTCTTGCTTGG 1 1 gbs0910gbs0911 ACATACTTTTAAAAGCATTATCTTTATCTTTCATCTGTTATTTGTTAAAATATCCTAAA 1 1 gbs0968 ACATACAAAATGTCTATTTAAATAGACATTTTTATTTTTGTCTGCTATAATAGACAAAA 2 1 3 gbs1031 ACATACTAAATTGGAAAAAAGATAGTCAAACTCTTTAATTTTTTGTATAATAGTCTCTA 1 1 gbs1082 pyrF ACATACATAATTTAGTATTTATTTGACAAAATGATTGTAAACTTGTAGAATACCTACAA 1 1 1 3 gbs1111 ACATACGCCATTGAAAACAGTGTGTACTTTTTCACAACATTATGTTACAATGATGCCCA 1 1 gbs1118gbs1117 ACATACTTCCTTTGCTTCAGTCTTGACCCTATTGCGGTTTTTATGCTACAATACTGTTT 1 1 gbs1223 ACATACATATCAGTTTAGTTAGTTGACAATAATAATAATATAATATAAAATGAAGATAA 1 1 gbs1232 ACATACATTATTCATCTTATTTTTGCGATTTCAATAGTGATATGGTATATTTAAAGTAA 1 1 gbs1308 scpB ACATACTTTTGCATGTTTTAACTTGCATTAAATCTTCTAAAGTAGTATAATTATATTAA 1 1 gbs1314 int ACATACCAAAGTTTGAAGAAACTAGTAATTCTACAAATAATTTGGTAAAATTGAGGAAA 2 2 gbs1385 ribD ACATACAAGTCTTGTTTCTCACTAGAATATCTTTTAAAAATCTGATAAAATAAGACTTT 1 1 gbs1468 rpoB ACATACAAAGACTAAGATTACTTAGTCTTTTTTTCATGAAAATGATATAATAAAGTCAT 1 1 gbs1497 dnaG ACATACAAGCTTTTGAAAAAATTTACACAGAAAAGAAAAAATGATATAATCAAATGTAA 1 1 gbs1499 rpsU ACATACAAGTATCATAAAAATATTGACAATCTGCTTTTATTTGGGTATCCTAGTAAGTG 12 1 2 15 gbs1626 tmk ACATACACAATCAAGTTGTGGGTGTCTTTTTTTGATAAATTTTGATAGAATAGTAGTAA 1 1 gbs1637 cspR ACATACAAAACTCATGAATAGGTTGACTTTGTAGACTGAAGTGCTATAATAGTGTCAAC 1 1 gbs1638 ACATACAATAACACAGATAAACTTGTGACAAGCCCTAAATTAGGTTATAATTAACCTTT 1 1 gbs1650 ACATACTTTTGTTTACATATATTTGTAAACTCTTTCTTTTTTTGCTATAATGAGTCATG 1 1 gbs1662 ACATACCATTGTCAAAGTTTTGTTGATTTTATAGACTATTTAAGATATAATTGCCTGTG 1 1 gbs1681 ACATACCTTCTTGTCAATAAGGTTTCAAATCAGCTTGAAATATGATAAAATAAAACAGA 1 1 gbs1691 ACATACATTTCCATTTTACCAATTGCTATCAAAGTGCTATAATAATAGAGTTAAAATTA 1 1 2 gbs1724 aroD ACATACTAAATTACATACTCTCTTTTTAATTGAGTCAGTTTTTGGTAAAATGAACTGAA 1 1 gbs1739 efp ACATACCAAATCAATAAACTATTTGAGAAAGCAGGTTATTTGTGATAAAATGAGTTGAT 1 1 2 gbs1776 ACATACATACCATAAAGTCAAATTGACAGTTTCACTATTTTTTTGTAGACTGGATATTA 1 1 gbs1777 glpF ACATACTTATTTTGTGATAAGCTTTACAATATTAAGCTTACTTGGTAGAATGGTTAATG 1 1 gbs1799 ACATACCATAATATCAAAACGCTTGTTTTGATATTTTTTATTTGGTATAATAATTGAAT 1 1 gbs1814 rpsL ACATACTAAAAGTAATAAAAGCTTGACACATCATGTGTAAAATGATACTATGATAAACG 1 1 2 gbs1835 dltR ACATACATAATGCGGAAATTCGTAGACAAATAGCCCCTATTTTTGTTAAAATATGAGGA 1 1 gbs1836 rpmH ACATACAACTTGAAAGTATTTCTTGACAAGAGGCCTAAAATTGTTATAAAATAGTATGG 1 1 2 gbs1849 ACATACCCAATAAGAACACTTATTTTCATTTTTCTTATTAAATGATATAATACGGATAA 1 1 2 gbs1928 relA ACATACTTGAGCCTCTAACTTCTTTTATTTTTAGGTTAAAAAGTGATAAAATAGGTACA 1 1 gbs2022 ACATACTTGAAATCGGATTATGGTTTCAATCCTTTTTTCGTTTTGTTATAATAGAAAAA 1 1 gbs2033 ACATACAAATCAAATTTAACTATTGACAATTATTTTTAAATGACGTAAACTTATAGGCA 1 1 gbs2101 gidA ACATACAGCCTATCCCCTCTGGTTGACTTTTAAAAGATAAATGCTAAAATAAATTTAAC 1 1 rRNA 5S ACATACTATCCAAAAAGAGATATTGACAACGTTACGGTTTCTTGTTAGACTATAGATAT 2 2 rRNA 16S ACATACACTTAAAAAAAAGTAGTTGACAAAGCAGAATAAGTTTGCTAGAATATAGAAGT 1 1 tRNA thr ACATACATTTCTCCAAATAGAATTAGACTAAAAAAAACGGCTATTTTTCTAAGTAAAGA 2 2 tRNA cys ACATACATTTTACGAAAGAGGCTTGACGAATCTTTTTTTTTTCGATAGAATATTATCTG 1 1

total 61 23 16 100

IR‐right ‐35 box ‐10 box

Figure S7

RepA_N

Rep_3

RepE

IntegraAon

Tyrosine recombinase

ReplicaAon

DDE transposase

Figure S8

modular evolution of tngbss, a new family of ices ......! 1! modular evolution of tngbss, a new...

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