Neo-formation of chromosomes in bacteria.

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Neo-formation of chromosomes in bacteria.

Olivier Poirion, Bénédicte Lafay

To cite this version:

Olivier Poirion, Bénédicte Lafay. Neo-formation of chromosomes in bacteria.. 2019. �hal-02402186�

Neo-formation of chromosomes in bacteria

1

Olivier B. Poirion

^1,2†

& Bénédicte Lafay

^1,2,3*

2

1

Université de Lyon, F-69134 Lyon, France 3

2

CNRS (French National Center for Scientific Research) UMR5005, Laboratoire 4

Ampère, École Centrale de Lyon, 36 avenue Guy de Collongue, 69134 Écully, France 5

3

CNRS (French National Center for Scientific Research) UMR5558, Laboratoire de 6

Biométrie et Biologie Évolutive, Université Claude Bernard – Lyon 1, 43 boulevard 7

du 11 novembre 1918, 69622 Villeurbanne cedex, France 8

†

Current address: Center for Epigenomics, Department of Cellular and Molecular 9

Medicine, University of California, San Diego, School of Medicine, 9500 Gilman Drive, 10

La Jolla, CA 92093, USA 11

* Author for correspondence: benedicte.lafay@univ-lyon1.fr 12

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A BSTRACT 13

Although the bacterial secondary chromosomes/megaplasmids/chromids, first noticed 14

about forty years ago, are commonly held to originate from stabilized plasmids, their 15

true nature and definition are yet to be resolved. On the premise that the integration of a 16

replicon within the cell cycle is key to deciphering its essential nature, we show that the 17

content in genes involved in the replication, partition and segregation of the replicons 18

and in the cell cycle discriminates the bacterial replicons into chromosomes, plasmids, 19

and another class of essential genomic elements that function as chromosomes. These 20

latter do not derive directly from plasmids. Rather, they arise from the fission of a multi- 21

replicon molecule corresponding to the co-integrated and rearranged ancestral 22

chromosome and plasmid. All essential replicons in a distributed genome are thus neo- 23

chromosomes. Having a distributed genome appears to extend and accelerate the 24

exploration of the bacterial genome evolutionary landscape, producing complex 25

regulation and leading to novel eco-phenotypes and species diversification.

26

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I NTRODUCTION 27

Chromosomes are the only components of the genome that encode the necessary 28

information for replication and life of the cell/organism under normal growth conditions.

29

Their number varies across taxa, a single chromosome being the standard in bacteria 30

(Krawiec and Riley, 1990). Evidence accumulated over the past forty years is proving 31

otherwise: bacterial genomes can be distributed on more than one chromosome-like 32

autonomously replicating genomic element (replicon) (Casjens, 1998; diCenzo and 33

Finan, 2017; Mackenzie et al., 2004). The largest, primary, essential replicon (ER) in a 34

multipartite genome corresponds to a bona fide chromosome and the additional, 35

secondary, ERs (SERs) are expected to derive from accessory replicons (plasmids 36

(Lederberg, 1998)). The most popular model of SER formation posits that a plasmid 37

acquired by a mono-chromosome progenitor bacterium is stabilized in the genome 38

through the transfer from the chromosome of genes essential to the cell viability 39

(diCenzo and Finan, 2017; diCenzo et al., 2013; Slater et al., 2009). The existence in 40

SERs of plasmid-like replication and partition systems (Dubarry et al., 2006; Egan and 41

Waldor, 2003; Livny et al., 2007; MacLellan et al., 2004, 2006; Slater et al., 2009;

42

Yamaichi et al., 2007) as well as experimental results (diCenzo et al., 2014) support this 43

view. Yet, the duplication and maintenance processes of SERs contrast with the typical 44

behaviour of plasmids for which both the timing of replication initiation and the 45

centromere movement are random (Million-Weaver and Camps, 2014; Reyes-Lamothe 46

et al., 2014). Indeed, the SERs share many characteristic features with chromosomes:

47

enrichment in Dam methylation sites of the replication origin (Egan and Waldor, 2003;

48

Gerding et al., 2015), presence of initiator titration sites (Egan and Waldor, 2003;

49

Venkova-Canova and Chattoraj, 2011), synchronization of the replication with the cell 50

cycle (De Nisco et al., 2014; Deghelt et al., 2014; Egan and Waldor, 2003; Egan et al., 51

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2004; Fiebig et al., 2006; Frage et al., 2016; Kahng and Shapiro, 2003; Rasmussen et al., 52

2007; Srivastava et al., 2006; Stokke et al., 2011), KOPS-guided FtsK translocation (Val 53

et al., 2008), FtsK-dependent dimer resolution system (Val et al., 2008), MatP/matS 54

macrodomain organisation system (Demarre et al., 2014), and similar fine-scale 55

segregation dynamics (Fiebig et al., 2006; Frage et al., 2016). Within a multipartite 56

genome, the replication of the chromosome and that of the SER(s) are initiated at 57

different time points (De Nisco et al., 2014; Deghelt et al., 2014; Fiebig et al., 2006;

58

Frage et al., 2016; Rasmussen et al., 2007; Srivastava et al., 2006; Stokke et al., 2011), 59

and use replicon-specific systems (Drevinek et al., 2008; Egan and Waldor, 2003;

60

Galardini et al., 2013; MacLellan et al., 2004, 2006; Slater et al., 2009). Yet, they are 61

coordinated, hence maintaining the genome stoichiometry (Deghelt et al., 2014; Egan et 62

al., 2004; Fiebig et al., 2006; Frage et al., 2016; Stokke et al., 2011). In the few species 63

where this was studied, the replication of the SER is initiated after that of the 64

chromosome (De Nisco et al., 2014; Deghelt et al., 2014; Fiebig et al., 2006; Frage et al., 65

2016; Rasmussen et al., 2007; Srivastava, 2006; Stokke et al., 2011) under various 66

modalities. In the Vibrionaceae, the replication of a short region of the chromosome 67

licenses the SER duplication (Baek and Chattoraj, 2014; Kemter et al., 2018), and the 68

advancement of the SER replication and segregation triggers the divisome assembly 69

(Galli et al., 2016). In turn, the altering of the chromosome replication does not affect 70

the replication initiation control of the SER in α-proteobacterium Ensifer/Sinorhizobium 71

meliloti (Frage et al., 2016).

72

Beside the exploration of the replication/segregation mechanistic, studies of multipartite 73

genomes, targeting a single bacterial species or genus (diCenzo et al., 2013, 2014;

74

Dubarry et al., 2006; Mackenzie et al., 2004; Slater et al., 2009) or using a more 75

extensive set of taxa (diCenzo and Finan, 2017; Harrison et al., 2010), relied on 76

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inadequate (replicon size, nucleotide composition, coding of core essential genes for 77

growth and survival (diCenzo and Finan, 2017; Harrison et al., 2010; Liu et al., 2015);

78

Figure 1) and/or oriented (presence of plasmid-type systems for genome maintenance 79

and replication initiation (Harrison et al., 2010)) criteria to characterize the SERs.

80

81

Figure 1. Structural features of the replicons 82

Boxplots of the lengths (base pairs) and numbers of genes (ORFs), protein-coding genes (CDS), pseudogenes,

83

ribosomal RNA genes and transfer RNA genes for the 2016 chromosomes (blue), 129 SERs (orange), and 2783

84

plasmids (green) included in the final dataset (4928 replicons).

85

10M

1 10 100 1000 10k 100k 1M

Log-transformed numberper replicon

Size (bp) ORFs CDS

120

0 20 40 60 80 100

Number per replicon

140 160

rRNA genes tRNA genes 0

200 400 600 800 1000

Number per replicon

Peudogenes

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While clarifying the functional and evolutionary contributions of each type of replicon 86

to a multipartite genome in given bacterial lineages (Galardini et al., 2013; Harrison et 87

al., 2010; MacLellan et al., 2004; Slater et al., 2009), these studies produced no absolute 88

definition of SERs (diCenzo and Finan, 2017; Harrison et al., 2010) or universal model 89

for their emergence (diCenzo and Finan, 2017; diCenzo et al., 2013, 2014; Galardini et 90

al., 2013; Harrison et al., 2010). We thus set out investigating the nature(s) and origin(s) 91

of these replicons using as few assumptions as possible.

92

R ESULTS

93

Replicon inheritance systems as diagnostic features 94

We did not limit our study to a particular multipartite genome or a unique gene family.

95

Rather, we performed a global analysis encompassing all bacterial replicons whose 96

complete sequence was available in public sequence databases (Figure 2). We reasoned 97

that the key property discriminating the chromosomes from the plasmids is their 98

transmission from mother to daughter cells during the bacterial cell cycle. The functions 99

involved in the replication, partition and maintenance of a replicon, i.e., its inheritance 100

systems (ISs), thence are expected to reflect the replicon degree of integration into the 101

host cycle.

102

We first faced the challenge of identifying all IS functional homologues. The inheritance 103

of genetic information requires functionally diverse and heterogeneous actuators 104

depending on the replicon type and the characteristics of the organism. Also, selecting 105

sequence orthologues whilst avoiding false positives (e.g., sequence paralogues) can be 106

tricky since remote sequence homology most likely prevails among 107

chromosome/plasmid protein-homologue pairs.

108

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bioRxiv preprint first posted online Feb. 13, 2018;

Statistical + biological evaluation

UNSUPERVISEDMODEL Structuration = fstructure(V)+error

MODEL:

replicon stabilisation = f(IS) + error

4928 replicon vectors SUPERVISEDMODEL Prediction = fstructure({Vf ,prediction}training,Vf)+error

ACLAMEKEGGRefSeq Query set 47,604 proteins5125 replicons 6,903,452 proteins 358,624 sequence homologues

BLAST annotation

92 families71 groups 127,853 Pfam domains 1,174,018 Pfam domains

HMMER HMMER TRIBE-MCL 7013 clustersQuality assessment 6096 clusters Ci 267,497 proteins 117 functionalities fi

917 noisy clusters 91,127 proteins

Cleaning

4928 replicon vectors Best BLAST hit

vr=(NC1r,...,NC6069r)

197  IS-null replicons vfr=(Nf1r,...,Nf117r) Supervised classification

Visual exploration Bipartite graph SER-SPECIFICFUNCTIONALITIESVfR=(vfr1,...,vf|R|) vfg=(Nf1g r∈g

∑

,...,Nfng r∈g

∑

)VfG =(vfg1,...,vf|G| )with

VR =(vr1,...,v|R| ) PCA + WARD ERT

INFOMAP SERIDENTIFICATION

Regression

Logit

Unsupervised classification

Fi gu re 2 . An al yt ic al p ro ce d u re

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Starting from an initial dataset of 5125 replicons, we identified 358,624 putative IS 110

functional homologues, overall corresponding to 1711 Pfam functional domains (Figure 111

3a), using a query set of 47,604 chromosomal and plasmidic IS-related proteins selected 112

from the KEGG and ACLAME databases (Tables 1 and 2).

113

Table 1. ACLAME families used in the building of the query set 114

P

ROCESS

F

AMILY

P

ROTEIN DESCRIPTION

Replication

32

RepB, pi, initiator protein, RepE, RepA

76

Rep, RepB, Rep of rolling circle initiator, RepA, RepU, OrfB, Rep2

107

RepC, RepCa1, RepCa2, RepCd

114

Helicase, UrvD rep helicase, helicase super family 1, Yga2F, helicase II

118

CdsE, CdsJ

133

RepA, W0005, RepA1/A2

171

RepA, RepB, putative theta replicative protein

207

replicative DNA helicase, DnaB, pGP1

208

RepA, W0013, W0041, RepFIB

224

long form TrfA, TrfA1, TrfA2, S-TrfA, plasmid initiation protein

237

RepA, putative RepA, truncated RepA

244

RepA, RepB, CopB, repA1/A2, w0004

294

Rop regulatory protein, RNAI modulator, RNA modulator, plasmid copy number control

297

primase activity/DNA initiation, LtrC/LtrC-like hypothetical protein, PcfD

330

DNA repair/ DNA helicase, type III restriction enzyme, res subunit, DEAD/DEAH box helicase

377

replicase, replication initiation, RepC, RepJ, RepE, RepL

383

RepA, Rb100

404

RepA,RepB,RepW

412

Rep, RepA

423

truncated RCR replication, RepRC, RepB, OrfA

426

cell division control protein 6 homolog

440

Rep 14-4, rm protein, RepA hypothetical protein

451

RepA, host type : Corynebacterium

477

Rep, RepS, RepE, host type : Bacillus, RepS, RepR

612

RepL, replication initiation

775

DNA helicase activity, RepA, putative helicase

854

DNA helicase activity, RepC, putative initiator protein

921

RepA

931

DNA replication initiation, putative protein, CdsD

1005

helicase activity, putative protein, hypothetical helicase

1055

RNA polymerase σ factor, σ 70 family, bacteriocin uviA, sigF/V/G, tetR, host type : Clostridium

1095

DNA repair/helicase, RuvB, DNA pol III γ and τ subunits, DNA pol δ subunit

1099

putative theta replicase, RepB, Rep2

1187

DNA replication, RepH, RepI

1288

RepA

1345

DNA primase activity, DNA primase , primase CHC2 family

1398

helicase activity, GcrE, GcrC

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1652

DNA repair/exonuclease activity, DNA exonuclease protein, SbcCD related protein

1837

putative replication protein

2881

RepC-like, Pif

Partition

4

plasmid partition protein, ParA, ParA IncC protein, ParA InC1/ IncC2, SopA, virC1

14

RepB, RepB partitionning, KorB repressor and partitionning, ParB-like domain, YefA, YdeB, ParB, ParB-like

102

DNA binding, partitionning protein, control protein, ParB, VirB, partition protein B

128

DNA segregation/DNA translocase activity, cell division FtsK/ SpoIIIE, SpoI, TraB

289

ParM family, go : translocase, hypothetical protein, rode shape protein, putative ATPase of class HSP70

316

microfilament motor activity, ParM family, StbA protein, stable inheritance protein, ParA

318

ATPase, regulation of cell division, chromosome patition, GumC, ExoP related protein, EpsB, MPA1 family

427

ATPase family, ParR family, ParB, StbB, mediator of plasmid stability

875

DNA binding, partitionning protein family ParB/Spo0J, YPMT1.28c

876

DNA binding, partitionning protein family ParB/Spo0J, YPMT1.29c

983

DNA binding, ParB, CopG

1227

DNA plasmid copy number control, CopG

2158

RepC

2894

DNA binding

Dimer resolution

5

serine based recombinase activity, ylb, resolvase, second invertase, TniR, ParA

10

tyrosine-based recombinase, integrase, putative integrase, Xer, recombinase-like SAM

101

plasmid dimer resolution, tyrosine-based recombinase, yld, SAM-like protein

170

tyrosine-based recombinase, OrfA, hypothetical protein

589

tyrosine based protein, Fis protein

688

tyrosine based protein, SAM like protein, XerD

Maintenance

100

Postsegregational killing system vapBC/vag

136

Postsegregational killing system parDE

156

Postsegregational killing system epsilon-zeta

201

Postsegregational killing system higBA

212

Postsegregational killing system parDE

293

Postsegregational killing system mazEF

319

Postsegregational killing system relBE

326

Postsegregational killing system mazEF

335

Postsegregational killing system HOK/SOK

338

Postsegregational killing system parDE

356

Postsegregational killing system parDE

366

Postsegregational killing system vapBC/vag

380

Postsegregational killing system phD-doc

428

Postsegregational killing system ccd

470

Postsegregational killing system yacA

474

Postsegregational killing system relBE

515

Postsegregational killing system relBE

556

Postsegregational killing system higBA

563

Postsegregational killing system ccd

588

Postsegregational killing system higBA

677

Postsegregational killing system higBA

798

Postsegregational killing system mazEF

916

Postsegregational killing system relBE

1031

Postsegregational killing system HOK/SOK

1180

Postsegregational killing system vapXD

1308

Postsegregational killing system HicAB

1559

Postsegregational killing system epsilon-zeta

1927

Postsegregational killing system mazEF

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3357

Postsegregational killing system, plasmid maintenance

4776

Postsegregational killing system, parC

4777

Postsegregational killing system parDE, parD

16584

Postsegregational killing system vapXD

Table 2. KEGG “Prokaryotic-type chromosome” orthology groups used in the building of the 115

query set 116

BRITE

HIERARCHY

KEGG

E

NTRY

N

AME

D

EFINITION

Chromosome replication

Initiation factors (bacterial)

K02313

^DnaA chromosomal replication initiator protein

K02314

DnaB replicative DNA helicase [EC:3.6.4.12]

K03346

DnaB2, DnaB replication initiation and membrane attachment protein

K02315

DnaC DNA replication factor, helicase loader

K02316

DnaG DNA primase [EC:2.7.7.-]

K11144

DnaI primosomal protein DnaI

K05787

HupA DNA-binding protein HU-alpha

K03530

hupB DNA-binding protein HU-beta

K04764

IhfA, HimA integration host factor subunit alpha

K05788

IhfB, HimD integration host factor subunit beta

K03111

ssb single-strand DNA-binding protein Terminus site-binding

protein

K10748

Tus, Tau DNA replication terminus site-binding protein

DNA methylation enzymeK06223 Dam DNA adenine methylase [EC:2.1.1.72]

Prevention of re- replication factors

K10763 Hda DnaA-homolog protein

K03645 SeqA negative modulator of initiation of replication

Chromosome partition

MukBEF complex

K03632 MukB chromosome partition protein MukB

K03804 MukE chromosome partition protein MukE

K03633 MukF chromosome partition protein MukF

Condensin-like complex

K03529 Smc chromosome segregation protein

K05896 ScpA segregation and condensation protein A

K06024 ScpB segregation and condensation protein B

Divisome proteins

K03585 AcrA membrane fusion protein

K01448

AmiA,AmiB,

AmiC N-acetylmuramoyl-L-alanine amidase [EC:3.5.1.28]

K13052

DivIC, DivA cell division protein DivIC

K03590

FtsA cell division protein FtsA

K05589

^FtsB cell division protein FtsB

K09812

FtsE cell division transport system ATP-binding protein

K03587

FtsI cell division protein FtsI [EC:2.4.1.129]

K03466

FtsK, SpoIIIE DNA segregation ATPase FtsK/SpoIIIE, S-DNA-T family

K03586

FtsL cell division protein FtsL

K03591

^FtsN cell division protein FtsN

K03589

FtsQ cell division protein FtsQ

K03588

FtsW, SpoVE cell division protein FtsW

K09811

FtsX cell division transport system permease protein

K03531

FtsZ cell division protein FtsZ

K09888

^ZapA cell division protein ZapA

K03528

ZipA cell division protein ZipA

Inhibitors of FtsZ assembly

K04074

^DivIVA cell division initiation protein

K06286

EzrA septation ring formation regulator All rights reserved. No reuse allowed without permission.

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K03610

^MinC septum site-determining protein MinC

K03609

MinD septum site-determining protein MinD

K03608

^MinE cell division topological specificity factor

K05501

SlmA, Ttk TetR/AcrR family transcriptional regulator

K09772

SepF cell division inhibitor SepF

K13053

^SulA cell division inhibitor, FtsZ assembly inhibitor

Other chromosome partitioning proteins

K04095

Fic cell filamentation protein

K04094

Gid, TrmFO methylenetetrahydrofolate--tRNA-[uracil-5-)-methyltransferase [EC:2.1.1.74]

K03495

GidA, MnmG,

MTO1 tRNA uridine 5-carboxymethylaminomethyl modification enzyme

K03501

GidB, RsmG 16S rRNA [guanine527-N7)-methyltransferase [EC:2.1.1.170]

K03569

MreB rod shape-determining protein MreB and related proteins

K03570

^MreC rod shape-determining protein MreC

K03571

MreD rod shape-determining protein MreD

K03593

^Mrp ATP-binding protein involved in chromosome partitioning

K03496

ParA, Soj chromosome partitioning protein

K03497

ParB, Spo0J chromosome partitioning protein, ParB family

K02621

ParC topoisomerase IV subunit A [EC:5.99.1.-]

K02622

ParE topoisomerase IV subunit B [EC:5.99.1.-]

K11686

^RacA chromosome-anchoring protein RacA

K05837

RodA, MrdB rod shape determining protein RodA

K03645

^SeqA negative modulator of initiation of replication

K03733

XerC integrase/recombinase XerC

K04763

XerD integrase/recombinase XerD

Nucleoid

HNS (histone-like nucleoid structuring

protein)

K03746

^H-NS DNA-binding protein H-NS

K11685

StpA DNA-binding protein StpA

HU (heat unstable protein)

K05787

^HupA DNA-binding protein HU-alpha

K03530

HupB DNA-binding protein HU-beta

IHF (integration host factor)

K04764

IhfA, HimA integration host factor subunit alpha

K05788

IhfB, HimD integration host factor subunit beta

Other nucleoid associated proteins

K05516

CbpA curved DNA-binding protein

K12961

DiaA chromosomal replication initiator protein

K02313

DnaA DnaA initiator-associating protein

K04047

Dps starvation-inducible DNA-binding protein

K03557

Fis Fis family transcriptional regulator, factor for inversion stimulation protein

K03666

^Hfq host factor-I protein

K05596

IciA chromosome initiation inhibitor, LysR family transcriptional regulator

K03719

Lrp leucine-responsive regulatory protein, Lrp/AsnC family transcriptional regulator

K05804

^Rob right origin-binding protein, AraC family transcriptional regulator All rights reserved. No reuse allowed without permission.

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117

Figure 3. Properties of the IS clustering 118

(a) Frequency distribution of the 358,624 putative IS protein homologues according to their number of functional domains (0 to

119

69) per protein (left), and occurrences of the 1711 functional Pfam domains (right). The 20 top most frequently encountered

120

functional domains are indicated. (b) Size distribution of the 7013 clusters, each comprising from a single to 1990 proteins. (c)

121

Percentage distribution of the most frequent annotation per cluster among all clusters (left) and among clusters with multiple

122

annotations (right). (d) Distribution of the most frequent annotation per cluster among the 917 excluded clusters (left) and the

123

6096 clusters retained for the analysis (right).

124

FtsE

Rob

AmiA, AmiB, AmiC

XerD IciA AcrA XerC

ParA, Soj Xer-like tyrosine

recombinase ParB, Spo0J Lrp ParA/ParM serine recombinase

CbpA Helicase

RuvB HupB

MreD FtsQ MinD FtsK, SpoIIIE FtsI DnaC Ssb

ATPase, TyrK, ExoP DNA helicase RepA,E,B SlmA, Ttk DnaG Mrp Smc ZapA Dam DnaB FtsX ParB MreC other FtsE

AmiA, AmiB, AmiC FtsK, SpoIIIE

MinD Smc GidB, RsmG ParB, Spo0J Mrp

FtsX DnaB ScpA ParA, Soj ZipA other

0 1000 2000 3000 4000 5000 6000 7000

1-5% 20-25% 45-50% 70-75% 95-100%

Number of clusters

% of the most frequent annotation per cluster 0 20 40 60 80 100 120

1-5% 20-25% 45-50% 70-75% 95-100%

Number of clusters with multiple annotations

% of the most frequent annotation per cluster

b c

d

6096 retained clusters 917 excluded clusters

0 20 000 40 000 60 000 80 000 100 000 120 000 140 000 160 000 180 000

2OG-FeII_Oxy 5_3_exonucA2M_comp AAA_10 AAA_15 AAA_2 AAA_25 AAA_3 AAA_35 ABC_membrane ABC_tran_2 ABC2_membrane_4 Abhydrolase_6 Acetyltransf_7 Actin Acyltransferase AFG1_ATPase Aldedh AlkA_N Amidase Amidase02_C AMP-binding_C_2 Ank_2 Anticodon_1 Apc4_WD40 Arabinose_bd Arch_ATPase Arm AsmAATG16 ATP-grasp_3 ATPgrasp_Ter B-block_TFIIIC Bac_DnaA_C BcrAD_BadFG BetaGal_dom 4_5 Big_4 BiPBP_C Borrelia_orfABRCTC-term_anchor CALCOCO1 Carboxype

pD_reg Cast CbiQ CBM_20 CBM_X2 CCDC144C CDC48_2 CENP-C_C CHB_HEX_C Chrome_Resist CMAS CobT_C Collar

Coupl

e_hipA CPT CTDII Cu_amine_oxidN3 Cupin_3 CW_binding_1 Cytochrom_C Cytochrome-c551 DAO DcpS_C DDE_Tnp_IS66 DDRGK DinB_2 DLH DNA_gyraseB_C DNA_pol_B DNA_pol3_delta DNA_primase_S DnaB_2 DnaJ DPBB_1 DRP DUF1049 DUF1175 DUF1510 DUF1664 DUF1778 DUF187 DUF1989 DUF2232 DUF2442 DUF2730 DUF29 DUF3099 DUF3258 DUF3380 DUF3435 DUF3491 DUF3620 DUF3701 DUF3794 DUF3863 DUF4047 DUF4101 DUF4135 DUF4163 DUF4201 DUF4347 DUF4368 DUF4411 DUF4480 DUF488 DUF559 DUF736 DUF830 DUF927 DUF977 EAL EcoR124_C EFG_C Endonuc_subdomERAP1_C Esterase_phd EzrA FAD_binding_3 Fascin

FeoB_N

Fer4_12 Fer4_17 Fer4_6 Ferritin FGGY_N Fic_N FixG_C FliJ Fn3_assoc Ftsk_gamma FTSW_RODA_SPOVE G5 GAS GATase_6 Germane GIDA_assoc_3 Gly_transf_sug Glyco_hydro_18 Glyco_hydro_20 Glyco_hydro_3 Glyco_hydro_39 Glyco_hydro_66 Glyco_hydro_cc Glyco_trans_1_2 Glyco_trans_4_4 Glycos_transf_2 GNVR GrpE Guanylate_cyc HAD HATPase_c HD HEAT_2 Helicase_C_3 Heme_oxygenase Hexapep_2 HHH_6 Hint_2 HisKAHlyD HNH HRDC HtaAHTH_15 HTH_21 HTH_26 HTH_30 HTH_37 HTH_5

HTH_AsnC-type HTH_Mga HU-DNA_bdg Hydrolase HYR IFT57 IncA Integrase_AP2 Jacalin KdpD Kelch_6 Kinase-like L_lactis_RepB_C LacY_symp Laminin_G_3 Lectin_legB Lipase_GDSL_2 Lon_2 LRR_1 LRR_9 Lyase_8_C Lysozyme_like LZ_Tnp_IS66 MAD MbeD_MobD MDMPI_N MerR Metallopep Methyltransf_11 Methyltransf_1NMethyltransf_26 Methyltransf_4 MFS_1 MgsA_C Miro MMR_HSR1 MoCF_biosynth MreB_Mbl MscS_porin MukE Mur_ligase_M MVIN NACHT NapD NERD Nitro_F

eMo-Co NodS NPV_P10 NTP_transf_5 NUMOD1 OEP OpcA_G6PD_assem P22_Coa tProtein ParA ParG PAS_8 PBP_dimer PD40 Pec_lyase PemK PepSY

Peptidase_C26 Peptidase_C69 Peptidase_M14 Peptidase_M20 Peptidase_M28 Peptidase_M6 Peptidase_S24 Peptidase_U35_2 Peripla_BP_4 PfkB PGI Phage_int_SAM_1 Phage_integ_NPhage_rep_O PhdYeFM_antitox PHP PIN Plasmid_stab_B PLDc_N PMT_2 Polysacc_deac_1 Porphobil_de amC PPC PQQ_3 Prenyltrans Pribosyltran Prim-Pol Propeptide_C25 PSD4 PT-TG Pyr_redox_2 Rad50_z

n_hook RCC1 Recombinase Reo_sigmaC Rep_trans RepC Response_reg Rhomboid Ribonuc_red_lgC Ribosomal_L22 Ribosomal_S30AE Rieske_2 RNA_helicase RNA_pol_Rpb1_5 RnfC_NRotamase_3 RQC

Rubre

rythrin rve S4 SbcD_C SBP_bac_5 SdrG_C_C Secretin SeqA SH3_2 SH3_9 Sigma54_activat Sigma70_r2 SIR2_2 SLT SNase Spc7 SpoU_methylase SSB Sugar_tr SurA_N_3 T2SS-T3SS_pil_N TBPIP Tektin Terminase_1 Terminase_GpA TetR_C_6 TGS ThiS Thymidylat_synt TIR_2 TOBE_3 Toprim_2 Toxi n_60 TPR_10 TPR_16 TPR_6 TraG-D_C Transglut_core3 Transpeptidase TrfA

tRNA_lig_kinase tRNA-synt_1e Trp_repressor Trypan_PARP Tubulin Ubie_methyltran UN_NPL4 UPF0114 UPF0547 UvrD_C VanW VirE VWA_3 WHG Y_phosphatase YadA_head YcgR_2 YHS YPEB Zeta_toxin zf-IS66 ZipA_C ZU5

Number of proteins

Pfam domain AAA_21

ABC transporter

SMC N

HTH Ara

C

HTH 1 LysR su

bstrate

Biotin lipoyl 2 ABC

me mb

rane

CbiA DNA gyra

se A C

oligo HPY

Ph age integrase AAAdoma

ins

HlyD doma

ins ABC domains

0 10 000 20 000 30 000 40 000 50 000 60 000 70 000 80 000

Number of proteins

Number of Pfam domains per protein

40 69

10

0 20 30 50 60

a

0 500 1 000 1 500 2 000 2 500 3 000 3 500 4 000 4 500 5 000

1-20 80-100 180-200 280-300 380-400 480-500 580-600 680-700 780-800 8980-900 980-1000 1080-1100 1180-1200 1280-1300 1380-1400 1480-1500 1580-1600 1680-1700 1780-1800 1880-1900 1980-2000

Number of clusters

Number of proteins per cluster (Cluster size)

We then inferred 7013 homology groups using a clustering procedure and named the 125

clusters after the most frequent annotation found among their proteins (Figure 3b,c). Most 126

clusters were characterized by a single annotation whilst the remaining few (4.7%) each 127

harbored from 2 to 710 annotations, the most frequent annotation in a cluster generally 128

representing more than half of all annotations (Figure 3c). The removal of false positives 129

left 267,497 IS protein homologues distributed in 6096 clusters (Figure 3d) and coded by 130

4928 replicons out of the initial replicon dataset. Following the Genbank/RefSeq 131

annotations, our final dataset comprised 2016 complete genome sets corresponding to 132

3592 replicons (2016 chromosomes, 129 SERs, and 1447 plasmids) and 1336 plasmid 133

genomes (Supplementary table 1), irregularly distributed across the bacterial phylogeny 134

(Figure 4a). Multi-ER genomes are observed in 5.0% of all represented bacterial genera 135

and constitute 5.7% of the complete genomes (averaged over genera) available at the time 136

of study (Figure 4b). They are merely incidental (0.2% in Firmicutes) or reach up to 137

almost one third of the genomes (30.1% in β-Proteobacteria) depending on the lineage, 138

and are yet to be observed in most bacterial phyla, possibly because of the poor 139

representation of some lineages. Although found in ten phyla, they occur more than once 140

per genus in only three of them: Bacteroidetes, Proteobacteria and Spirochaetae.

141

142 143

Fi gu re 4 . Ta xo n om ic s tr u ct u re o f th e re p li co n d at as et Nu m be rs o f re pl ic on s (a ) an d co m pl et e ge no m es ( b) , a nd r ep re se nt ed b ac te ri al g en er a ar e sh own a cc or di ng t o da ta se ts a nd h os t ta xo no m y. S ur fa ce s re pr es en t th e nu mb er s of b ac te ri al g en er a, r ep li co ns o r ge nom es w it hi n ea ch ca te gor y. P er ce nt age s of m ul ti pa rt it e ge nom es a nd cor re spon di ng ba ct er ia l g ene ra a re c al cu la te d for e ac h hos t ph yl um or c la ss ( P ro te oba ct er ia ).