supplementary information - nature · supplementary information supplementary figures. no:...

1

Supplementary Information Supplementary Figures.

Supplementary Figure 1. Purified mycobacterial NucS does not cleave mismatch DNA

substrates in vitro. A) Native M. smegmatis NucS protein purification. Recombinant NucS protein

was expressed in E. coli, purified and concentrated, as described (see Methods). Native NucS

protein was analysed in a SDS-PAGE gel and stained with Coomassie Brillant Blue. Lane 1,

molecular marker; Lane 2: purified native M. smegmatis NucS; Lane 3: concentrated M. smegmatis

NucS protein (used for EMSA and nuclease assays). B) Double stranded DNA (30 nM) with or

without the indicated internal mismatches, was incubated with 300 nM NucS. Sequence consensus

is shown at the bottom and mismatch positions are indicated in red. No significant cleavage activity

was observed with the shown 36-mers or with longer 75-mer (not displayed here) mismatch

substrates.

C-A:C-A:C-T:C-T:C-C:C-C:T-T:T-T:T-G:T-G:A-A:A-A:A-G:A-G:G-G:G-G:

NO:NO:

37

25

50

20NucS

15

75

1 2 3200

A B

10

kDa

150

100

dsDNA

2

A

B

Supplementary Figure 2. Tools for measuring recombination rates.

A) Alignment of the 517 bp overlapping sequences, with 100%, 95%, 90% and 85% identities,

shared by hyg 5´ and hyg 3´ regions, used to construct the different pRhomyco versions. Changes

3

introduced in the original sequence are highlighted. B) Cartoon of plasmid pRhomyco before and

after recombination. hyg 5’ and hyg 3’ are truncated alleles of the hyg gene carrying a 3´-terminal or

5´-terminal deletion, respectively. Both alleles overlap 517 bp (striped regions) and are separated by

a 1,200 bp region containing aph3 gene (Kan-R). Four versions of pRhomyco were generated

carrying hyg 5’ alleles 100%, 95%, 90% or 85% identical, in its overlapping region, to the hyg 3’.

Plasmids integrate in the chromosome of M. smegmatis mc2 155 and its ΔnucS derivative. Site-

specific recombination between the attP from the plasmid and the unique bacterial attB is promoted

by the plasmid-encoded integrase.

4

Supplementary Figure 3. Domain characterization of M. tuberculosis NucS and polymorphic

residues. A) The upper part is a schematic representation of the homodimeric structure of P. abyssi

NucS (2VDL) 1. The two distinct domains of NucS, N-terminal and C-terminal, are in blue and

N-terminal domainC-terminal domainA

G157

D160

E174

Q187

Y191

K176

R42

R70

W75

HGGVVTIFARCKVHYEGRAKSELGEGDRIIIIKPDGSFLIHQN-KKREPVNWQPPGSKVT2VLD_Paby 25

M---RLVIAQCTVDYVGRLTAHLPSARRLLLFKADGSVSVHADDRAYKPLNWMSPPCWVTNucS_Mycsme 1

MSRVRLVIAQCTVDYIGRLTAHLPSARRLLLFKADGSVSVHADDRAYKPLNWMSPPCWLTNucS_Myctu 1

* * * * * * * ** * * ** * * *

* * * *

* * *

* * * ** * *

** * * *

*

* * **

* * * *

* * *

**

FK--E--NSmISIRRRPYERLEVEIIEPYSLVVFLAEDYEELaltgSEAEmANLIFENPR2VLD_Paby 84

EQDTETGVALWVVENKTGEQLRITVEDIEHDSHHELGVDPGLVKDGVEAHLQALLAEHVENucS_Mycsme 58

EESGGQ-APVWVVENKAGEQLRITIEGIEHDSSHELGVDPGLVKDGVEAHLQALLAEHIQNucS_Myctu 61

VIEEGFKPIYREKPIRHGIVDVMGVDKDGNIVVLELKRRKADLHAVSQLKRYVDSLKEEY2VLD_Paby 140

LLGAGYTLVRREYPTPIGPVDLLCRDELGRSVAVEIKR-RGEIDGVEQLTRYLELLNRDSNucS_Mycsme 118

LLGEGYTLVRREYMTAIGPVDLLCRDERGGSVAVEIKR-RGEIDGVEQLTRYLELLNRDSNucS_Myctu 120

-GENVRGILVAPSLTEGAKKLLEKEGLEFRKLEPP2VLD_Paby 200 233

LLAPVAGVFAAQQIKPQARTLATDRGIRCVTLDYDNucS_Mycsme 177 211

VLAPVKGVFAAQQIKPQARILATDRGIRCLTLDYDNucS_Myctu 179 213

*

180o

Q187

K176

Y191

E174

Q187

A67V69

S54

S39

W75W75 R70R70

R42R42

A135

D162

Q187

K184

Y191

E127 D160

G157

A67

R144

K184

A135

D162

Q187

T168

B

* R I

S A

S S H A

E

R144

T168

S39

S54

V69

W75W75 R70R7

0

R42R42

*

E127

C-terminal domain

5

pink, respectively. The first residues (1-25) of the NucS P. abyssi N-terminal domain, missing in

the M. tuberculosis model, are depicted as green cartoon. The putative β-clamp binding motif in P.

abyssi NucS is shown in orange. The important catalytic and DNA binding sites are depicted over

the P. abyssi structure as vertical red bars with the residues numbered above. Back and front views

of the structural superimposition of the homodimeric resolved structure of NucS from P. abyssi 1

and the M. tuberculosis model (purple) is presented below the schema (colour code as described for

the upper scheme). The important catalytic and DNA binding sites are depicted as red sticks and the

residues where polymorphisms described in this work have been detected are shown as green sticks

over the structural imposition. B) Multiple structure-based alignment of NucS from P. abyssi

(2VDL), M. smegmatis mc2 155 and M. tuberculosis CDC155. Colour code is the same as in panel

A. The amino acid change of each naturally occurring M. tuberculosis polymorphism is depicted in

green. Important catalytic and DNA binding residues are in red. Grey low-case indicates regions

without structural information; magenta “m” indicates Seleno-Met modifications in the structure of

P. abyssi. Residues of the putative β-clamp binding motif in P. abyssi NucS are shown in orange.

Only regions that align among the three proteins are shown. Asterisks indicate identical residues in

the three aligned sequences.

6

Supplementary Figure 4. Computational procedures to analyse NucS distribution and

evolution. Each panel depicts a different protocol. Yellow fonts indicate figures and/or tables.

Yellow uppercase letters are pieces of evidence used in the model presented in Fig. 6 (main text).

Domain Analyses IInitial Sequence Searches

~400

Compare ranked positions hits by e-value and bit-score

NucS_pyrab NucS_myctu

e-val < 0.0001, aln lenght > 75%, Bit score > 50

- ~370 unique archaeal/bacterial proteins- Eukaryotes and Virus discarded as lack NucS (A)

Align sequences (excluding original queries)

pHMMER

MAFFT

hmmbuild

hmmsearche-val < 0.0001, aln lenght > 75%,

Bit score > 50

~400

370 370

External info

Input file

Output file

Results

Program

MutS/L identification

hmmbuild

hmmsearch

MutS MutL Selected species

Bit score > 50, aln lenght > 75%,

MUTSMutL seqs MutS seqs

pMutS pMutL2,841 prokaryotic

genomes

MAFFT

alnMutS alnMutL

Phylogenetic ProfilingFigure 5

Supplementary data file 1

Large DB including 3,942 Ref Proteomes

aln-nr 63%


p-aln p-aln

aln-nr 63%

Structure-based definition using NucS_pyrab:full, N-terminal and C-terminal.

ArchaeaBacteria

Archaea/BacteriaArchaea-others

Archaea-others

Archaea/BacteriaProkayotes-othersEukaryotes

EukaryotesMAFFT- hmmbuild

Archaea-NucSBacteria-NucS

MAFFT-hmmbuild

Archaea-NucSBacteria-NucS

hmmbuild

hmmsearch

NucS is built on two distinct domains (B) Supp. Fig 3


hmmsearch Large DB including 3,942 Ref Proteomes

p-NT p-CT

aFull aNT aCT

pFull pNT pCT

Tree of Life(A)

(B) (C)

(D)

Proposed evolution of NucSFigure 6

MutS/L

Extract PFAM PF01939 seqs

Domain Analyses II

hmmalign3D-based pairwise

RAXML: phylogeny

NucS- full sequence Phylogenetic tree

NucS in some bacteria have been transferred from Archaea (C)

aFull aNT aCT

Supp. Fig 5 Supp. Fig 7 Supp. Fig 6

NucS N-Terminal Phylogenetic tree

NucS C-Terminal Phylogenetic tree

NucS shows a disperse distribution pattern (D)

(*) Only complete genomes Actinobacteria phylum and Archaea** Confident absence

If NO NucS is identified

if MutS/L is found if MutS/L not found: and “Unassembled WGS” else, translated searches vs genome(*) if MutS/L found if MutS/L not found

If NucS IS identified if MutS/L is found if MutS/L not found: and “Unassembled WGS” else, translated searches vs genome(*) if MutS/L found if MutS/L not found

Undef MutS/L

MutS/LLikelyNot

NucS-MutS/L

NucS-MutS/LNucS-only**

Procedure:

Undef MutS/L

and “Unassembled WGS” Undef NucS else, translated searches vs genome(*) if NucS found if NucS not found

NucSLikelyNot

7

Supplementary Figure 5. Phylogenetic analyses of NucS. Unrooted ML tree of full NucS

sequences from the PFAM PF01939. Black branches are Actinobacteria; blue branches are

Archaea; red branches are species from the Deinococcus-Thermus group. Labels indicate species

name. Red circles represent >80% bootstrap in 1,500 replicates.

Methanocella_paludicola.st.D

SM_17711

Stre

ptom

yces

_sp.

_Tu6

071

Streptomyces_sp._W

M6386

09761.MS

D.ts.iybslaw_

mutardauqolaH

Ther

moc

occu

s_ga

mm

atol

eran

s.st

.DSM

_152

29

Actinomyces_sp._oral_taxon_848_str._F0332

Corynebacterium_epidermidicanis

Amycolicicoccus_subflavus.st.DSM_45089

Actinomyces_sp.oral_tax_180.str.F0310

Natronobacterium

_gregoryi_ATCC

.43098

Microbacte

rium_hyd

rocarbonoxyd

ans

Corynebacterium_jeikeium.st.K411

Aeropyrum_pernix_ATCC.700893

Halobacterium

_sp._DL1

Mycobacterium_abscessus_ATCC.19977

Halobacterium

_sp._DL1

Actinobacteria_bacterium_O

V450

Stre

ptom

yces

_sp.

_CNQ

-509

Corynebacterium_argentoratense_DSM.44202

Conexibacter_woesei.st.DSM_14684

Streptomyces_m

angrovisoli

Nocard

ia_cy

riacig

eorgi

ca.st

.GUH-2

Saccharomonospora_glauca_K62

Mycobacterium_parascrofulaceum_ATCC.BAA-614

Stre

ptom

yces

_sp.

_WM

6378

Gardnerella_vaginalis_ATCC.14019

Rhodococcus_jostii.st.RHA1

Pyro

bacu

lum

_aer

ophi

lum

_ATC

C.51

768

Stre

ptom

yces

_rub

ello

mur

inus

Actinomyces_massiliensis_F0489

Rhodococcus_aetherivorans

Mycobacterium_sinense.st.JDM601


Rhodococcus_sp._B7740

Rothia_mucilaginosa.st.DY-18

Geodermatophilus_obscurus_ATCC.25078

Thermom

onospora_curvata_ATCC.19995

Kutzneria_albida_DSM.43870

Pyro

cocc

us_f

urio

sus_

ATC

C.4

3587

Nocardiopsis_sp._NRRL_B-16309

Frigoribacterium_sp._RIT-PI-h

Stre

ptomyc

es_r

imos

us_s

ubsp

._rim

osus

_ATC

C.1097

0

Blastococcus_saxobsidens.st.DD2

Pseudonocardia_dioxanivorans_ATCC.55486

Met

hano

pyru

s_ka

ndle

ri.st

.AV1

9

Arthrobacter_globiformis.NBRC.12137

Streptomyces_sp._M

MG

1121

Trueperella_pyogenes

Mycobacterium_hassiacum_DSM.44199

Streptomyces_cattleya_ATCC.35852

Rhodococcus_equi_ATCC.33707

Corynebacterium_glucuronolyticum_ATCC.51866

Stre

ptom

yces

_tsu

kube

nsis

_NR

RL1

8488

Streptomyces_averm

itilis_ATCC

.31267

Actinotignum_schaalii

Kocuria_sp._UCD-OTCP

Actinobacteria_bacterium_O

V320

Saccharothrix_sp._NRRL_B-16348

Mycobacterium_paratuberculosis_ATCC.BAA-968

Natrinem

a_sp.st.J7-2

Halorubrum

_lacusprofundi_ATCC

.49239

Nocardia_nova

_SH22a

2290

07.

CCT

A_mu

ranil

as_

muir

etca

bola

H

Microbacte

rium_laevaniformans_OR221

Brevibacterium_mcbrellneri_ATCC.49030

Arthrobacter_arilaitensis.st.DSM_16368

Corynebacterium_glutamicum_ATCC.13032

Corynebacterium_resistens.DSM.45100

Pyro

cocc

us_a

byss

i.st.G

E5

Cellulomonas_gilvus_ATCC.13127

8005

.ts.

sisn

egna

ggnij

_.ps

bus_

suci

pocs

orgy

h_pe

rtS

Actinomyces_sp._oral_tax_175.str.F0384

Strepto

myces

_gris

eus_

subs

p._gr

iseus

.st.JC

M_462

6

Sulfo

lobu

s_so

lfata

ricus

_ATC

C.35

092

Beutenbergia_cavernae_ATCC.BAA-8

Stre

ptom

yces

_viri

doch

rom

ogen

es

Bifidobacterium_scardovii

Scardovia_wiggsiae_F0424

Marinitherm

us_hydrothermalis.st.DSM

.14884

Sanguibacter_keddieii_ATCC.51767

Mobiluncus_mulieris_ATCC.35239

Actin

obac

teria

_bac

teriu

m_I

MCC

2625

6Actinomyces_graevenitzii_C83

Streptomyces_sp.

Cellulomonas_fimi_ATCC.484

marine_actinobacterium_PHSC20C1

Kita

sato

spor

a_se

tae_

ATCC

.337

74

Actin

obac

teria

_bac

teriu

m_O

K006

Bifidobacterium_gallicum_DSM.20093

Stre

ptom

yces

_gla

uces

cens

Deinococcus_soli_Cha_et_al._2014

Haloterrigena_turkm

enica_ATCC

.51198

Stre

ptom

yces

_sp.

_769

Hal

ofer

ax_v

olca

nii_

ATC

C.2

9605

Streptomyces_sp._e14

Renibacterium_salmoninarum_ATCC.33209

Micr

oluna

tus_

phos

phov

orus

_ATC

C.70

0054

Saccharothrix_espanaensis_ATCC.51144

Dietzia

_cinn

amea

_P4

Deinococcus_proteolyticus_ATCC.35074

Microb

acter

ium_o

xyda

ns

Corynebacterium_vitaeruminis_DSM.20294

Nocard

ia_far

cinica

.st.IF

M_101

52

Nocardia_sp._NRRL_S-836

Mycobacterium_thermoresistibile_ATCC.19527

Corynebacterium_diphtheriae_ATCC.700971

Actinoplanes_sp._N902-109

Bifidobacterium_longum.st.NCC_2705

Stre

ptom

yces

_sp.

_NRR

L_F-

6602

Mycobacterium_haemophilum

Gordonia_polyisoprenivorans.st.DSM.44266

Pyrolobus_fumarii.DSM

.11204

Stre

ptom

yces

_sp.

_PB

H53

Bifidobacterium_bifidum_BGN4

Dermacoccus_nishinomiyaensis

misce_Crenarchaeota_group-1_archaeon_SG8-32-1

Stre

ptom

yces

_dav

awen

sis_

JCM

_491

3

Bifidobacterium_angulatum_DSM.20098

Stre

ptom

yces

_xia

men

ensis

Microbacte

rium_ketosire

ducens

Mycobacterium_vanbaalenii.st.DSM.251

Nocardioides_simplex

Corynebacterium_kutscheri

Met

hano

spha

era_

stad

tman

ae_A

TCC

.430

21

Stre

ptom

yces

_coe

licol

or_A

TCC

.BAA

Ilumatobacter_coccineus_YM16-304

Stre

ptom

yces

_sp.

_AS5

8

Mycobacterium_leprae.st.TN

Gordonia_sputi_NBRC.100414

Mobiluncus_curtisii_ATCC.43063

Mobilic

occu

s_pe

lagius

_NBRC.10

4925

Nakamurella_multipartita_ATCC.700099

Prop

ionim

icrob

ium_ly

mphop

hilum

_ACS-

093-

V-SC

H5

Met

hano

brev

ibac

ter._

AbM

4

Streptomyces_sp._M

g1

Clavibacter_michiganensis_subsp._sepedonicus_ATCC.33113

Halostagnicola_larsenii_XH

-48

Corynebacterium_atypicum

Nocardioides_luteus

Streptomyces_vietnam

ensis

Streptomyces_sp._N

RR

L_F-4428

archaeon_GW2011_AR10

3809

2.C

CTA_

suec

ivs_

secy

motp

ertS

Frankia_symbiont_subsp._Datisca_glomerata

Micromonospora_sp._NRRL_B-16802

Frankia_sp._EUN1f

Thermobifida_fusca.st.YX

Rothia_dentocariosa_ATCC.17931

Stre

ptom

yces

_var

iega

tus

misce_Crenarchaeota_group-6_archaeon_AD8-1

Streptomyces_scabiei.st.87.22

Janibacter_hoylei_PVAS-1

Microbacte

rium_trichothecenolyti

cum

Corynebacterium_pseudotuberculosis.st.C231

Strepto

myces

_nive

us_N

CIMB_1

1891

Stre

ptom

yces

_lydic

us_A

02

Ther

mop

rote

us_s

p._A

Z2

Kytococcus_sedentarius_ATCC.14392

Stre

ptom

yces

_sp.

_NRR

L_F-

6602

Mycobacterium_obuense

Bifidobacterium_longum_subsp.longum_F8

Xylanimonas_cellulosilytica.st.DSM_15894

Mycobacterium_tuberculosis_H37Rv

Cellulomonas_flavigena_ATCC.482

Stre

ptom

yces

_sp.

_NRR

L_S-

495

Arthrobacter_sp._PAMC25486

Micromonospora_sp._HK10

Streptomyces_caelestis

Cald

ivirg

a_m

aqui

linge

nsis_

ATCC

.700

844

Stre

ptom

yces

_gris

eofla

vus_

Tu40

00

Mycobacterium_elephantis

Deinococcus_gobiensis.st.DSM_21396

Corynebacterium_xerosis

Microbacte

rium_chocolatum

Mycobacterium_smegmatis_ATCC.700084

Stre

ptom

yces

_viri

doch

rom

ogen

es.s

t.DSM

_407

36

Natronobacterium

_gregoryi_ATCC

.43098

Verrucosispora_maris.st.AB-18-032

Streptomyces_sp._AA4

Segniliparus_

rotundus_ATCC.BAA-972

Jonesia_denitrificans_ATCC.14870

Gordonia_sihwensis

_NBRC.108236

69781.M

SD.ts.ilagtoej_succocilaklala

H

Corynebacterium_falsenii_DSM.44353

Rhodococcus_spRD6.2

Bifidobacterium_dentium_ATCC.27534

Bifidobacterium_thermophilum_RBL67H

alopiger_xanaduensis.st.DSM

.8323

Amycolatopsis_orientalis_HCCB10007

Curtobacterium_flaccumfaciens_UCD-AKU

Streptosporangium_roseum

_ATCC.12428

T911

CS

UM_

.ps_

secy

motp

ertS

Met

hano

brev

ibac

ter_

rum

inan

tium

_ATC

C.3

5063

Acidothermus_cellulolyticus_ATCC.43068

33704_M

SD.ts.sunilloc_secy

motpertS

Microb

acter

ium_s

p._SA39

Mycobacterium_kansasii_ATCC.12478

Arthrobacter_chlorophenolicus_ATCC.700700

Met

hano

cald

ococ

cus_

jann

asch

ii_AT

CC

.430

67

4768

1_M

SD.t

s.si

snep

aloo

m_sa

nomo

nort

aN

Leucobacter_sp._Ag1

Brach

ybac

terium

_sp._

SW01

06-0

9

Streptomyces_sp._N

RR

L_S-444

Saccharopolyspora_erythraea_ATCC.11635

Deinococcus_swuensis

Streptomyces_sp._NRRL_F-6491

Vulca

nisa

eta_

dist

ribut

a.st

.DSM

.144

29

Mycobacterium_xenopi_RIVM700367

Saccharomonospora_marina_XMU15

Bifidobacterium_asteroides.st.P

RL2011Th

erm

ococ

cus_

koda

kare

nsis

_ATC

C.B

AA

Prop

ioniba

cteriu

m_a

cnes

.st.K

PA17

1202

Stre

ptom

yces

_sp.

_PVA

_94-

07

Stre

pt_c

yane

ogris

eus_

subs

p.no

ncya

noge

nus

Micrococcus_luteus_ATCC.4698

Corynebacterium_mustelae

Janibacter_sp._HTCC2649

Rhodococcus_erythropolis.st.PR4

Streptomyces_sp._N

RR

L_WC

-3618

679.21_S

D_snaticso_.psbus_sunegomorhcoesor_tpert

S

Terrabacter_sp._28

Acidi

anus

_hos

pitali

s.st.W

1

Natrialba_m

agadii_ATCC

.43099

Bifidobacterium_adolescentis_ATCC.15703

Microb

acter

ium_fo

liorum

Catenulispora_acidiphila.st.DSM_44928

Microbacte

rium_aza

dirach

tae

Lechevalieria_aerocolonigenes

Amycolatopsis_mediterranei.st.S699

Corynebacterium_sp._ATCC.6931

Gordonia_rhizosphera_NBRC.16068

Thermosphaera_aggregans.st.D

SM.11486

Salinispora_tropica_ATCC.BAA-916

I4F2P8 acti 477641

Mycobacterium_marinum_ATCC.BAA-535

halophilic_archaeon_DL31

Streptomyces_katrae

Streptomyces_sp._W

M6372

Bifidobacterium_breve_DSM.20213

94034.C

CTA_iutromsira

m_alucraolaH

Actin

obac

teria

_bac

teriu

m_O

K074

Methanocella_arvoryzae.st.D

SM.2066

Thermofilum_sp._1910b

Streptomyces_him

astatinicus_ATCC.53653

Tsuk

amure

lla_p

aurom

etabo

la_ATCC.83

68

Thermofilum

_pendens.st.Hrk_5

Corynebacterium_variabile.st.DSM_44702

Frankia_sp.st.EuI1c

Bifidobacterium_kashiwanohense_PV20-2

Deinococcus_maricopensis.st.DSM

.21211 Met

hano

ther

mob

acte

r_th

erm

auto

troph

icus

_ATC

C.2

9096

Prop

ioniba

cteriu

m_acid

iprop

ionici

_ATC

C.4875

Gordonia_amarae_NBRC.15530

Stre

ptom

yces

_inc

arna

tus

Mycobacterium_neoaurum_VKM_Ac-1815D

Actinoplanes_sp._ATCC.31044

Mycobacterium_sp.st.KMS)

Patulibacter_medicamentivorans

Nocardia_brasili

ensis_ATCC.700358

Strepto

myces

_prat

ensis

_ATC

C.3333

1

Nocardioides_sp.st.BAA-49

Thermogladius_cellulolyticus.st.1633

Hal

ogeo

met

ricum

_bor

inqu

ense

_ATC

C.7

0027

4

Corynebacterium_efficiens.st.DSM_44549

Igni

spha

era_

aggr

egan

s.st

.DSM

.172

30

Kineos

phae

ra_lim

osa_

NBRC.1003

40

Stre

ptomyc

es_p

ristin

aesp

iralis

_ATC

C.2548

6

Nocardioides_sp._CF8

Thermobispora_bispora_ATCC.19993

Staphylothermus_m

arinus_ATCC

.43588

Kocuria_rhizophila_ATCC.9341

Actinomyces_urogenitalis_DSM.15434

Corynebacterium_glyciniphilum_AJ_3170

Gordonia_sp._KTR9

Natronococcus_occultus_SP4

Frankia_sp.st.EAN1pec

Met

hano

bact

eriu

m_s

p._M

B1

Stre

ptom

yces

_nod

osus

Streptomyces_sp._M

MG

1533


Streptomyces_bingchenggensis.st.BCW

-1

Hyperthermus_butylicus.st.DSM

_5456

Actinosynnema_mirum_ATCC.29888

Prop

ioniba

cteriu

m_p

ropio

nicum

.st.F

0230

a

478007.C

CTA_ieatahokum_

muiborcimola

H

Streptomyces_pluripotens

Strepto

myces

_sp.s

t.Sire

xAA-E

Met

hano

brev

ibac

ter_

smith

ii_AT

CC

.350

61

Micromonospora_lupini_str._Lupac_08

Natronom

onas_pharaonis_ATCC

.35678

Met

hano

bact

eriu

m_l

acus

.st.A

L-21

Arcanobacterium_haemolyticum_ATCC.9345

Stre

ptom

yces

_ven

ezue

lae_

ATCC

.107

12

Natrinem

a_pellirubrum.st.D

SM_15624

Stre

ptom

yces

_aur

atus

_AGR0

001

Brach

ybac

terium

_faec

ium_A

TCC.43

885

Microbacte

rium_testa

ceum.st

.StLB037

L0J2N8 acti 212767

Stre

ptom

yces

_sp.

st.S

PB07

4

Actinoplanes_missouriensis_ATCC.14538

Actinomyces_turicensis_ACS-279-V-Col4

Mycobacterium_sp._VKM_Ac-1817D

Bifidobacterium_animalis_subsp._lactis.st.AD011

Streptomyces_sp._W

M4235

Gardnerella_vaginalis_JCP8481B

Austw

ickia_

chelo

nae_

NBRC.1052

00

Streptomyces_zinciresistens_K42

Arthrobacter_sp.st.FB24

Ther

moc

occu

s_lit

oral

is_A

TCC

.518

50

Streptom

yces_sp._MU

SC

136T

Kribbella_flavida.st.DSM_17836

Microb

acter

ium_s

p._Ag1

Aeromicrobium_marinum_DSM.15272

Leifsonia_xyli_subsp._xyli.st.CTCB07

Intrasporangium_calvum_ATCC.23552

Kineococcus_radiotolerans_ATCC.BAA-149

Sulfo

lobu

s_to

koda

ii.st.D

SM_1

6993

Microbacte

rium_ginsengisoli

Frankia_alni.st.ACN14a

Deinococcus_gobiensis.st.DSM_21396

Corynebacterium_matruchotii_ATCC.14266

Corynebacterium_urealyticum_ATCC.43042

Halobacterium

_salinarum_ATC

C.700922

Streptomyces_violaceusniger_Tu_4113

Gordonia_bronchialis_ATCC.25592

Bifidobacterium_pseudolongum_PV8-2

Stre

ptom

yces

_alb

us_A

TCC.

2183

8

Sulfo

lobu

s_ac

idoc

alda

rius_

ATCC

.339

09

Saccharomonospora_viridis_ATCC.15386

Strepto

myces

_clav

ulige

rus_A

TCC.2706

4

Frankia_sp.st.CcI3

Isoptericola_variabilis.st.225

540M_sucaitnaruaoesirg_secy

motpertS

Sacc

haro

thrix

_sp.

_ST-

888

Actinoplanes_friuliensis_DSM.7358

Mycobacterium_rhodesiae.st.NBB3

Stre

ptom

yces

_gha

naen

sis_

ATC

C.1

4672

Prop

ioniba

cteriu

m_f

reud

enre

ichii_

subs

p._s

herm

anii_

ATCC

.961

4

Nocardiopsis_alba_ATCC.BAA-2165

Candidatus_Caldiarchaeum_subterraneum

Turicella_otitidis_ATCC.51513

Rhodococcus_pyridinivorans_SB3094

Micromonospora_aurantiaca_ATCC.27029

Scardovia_inopinata_F0304

Met

hano

bact

eriu

m_p

alud

is.s

t.DSM

.258

20

Mycobacterium_sp._EPa45

Actinomyces_neuii_BVS029A5

Nocardiopsis_dassonvillei_ATCC.23218

Stackebrandtia_nassauensis.st.DSM_44728

Ther

moc

occu

s_ba

roph

ilus.

st.D

SM_1

1836

Tree scale: 0.1

ARCHAEA

ACTINOBACTERIA

Deinococcus

Tree scale

8

Supplementary Figure 6. Phylogenetic analyses of the NucS C-terminal (CT) region. Unrooted

ML tree of CT sequences from the PFAM PF01939 domain. Grey font indicates that this domain is

in NucS and blue font that is found outside NucS. Black branches, Actinobacteria; red

Deinococcus-Thermus; grey, other Bacteria; light blue, Archaea; green, eukaryotic sequences. Bold

fonts indicate the main species used in this study. Labels indicate species name. The circles

represent >80% bootstrap in 1,500 replicates.

111-

Actin

obac

teria

bac

teriu

m O

V320

415-Microbacterium ketosireducens

31202.MS

D.everb muiretcabodifiB-803

35-Methanocella arvoryzae.st.DSM 22066

348-

Marini

therm

us hy

droth

ermali

s.st.D

SM 14

884

169-Corynebacterium camporealensis

0790

1.C

CTA

suso

mir.p

sbus

sus

omir

sec

ymo

tpert

S-39

249-Gordonia sihwensis NBRC 108236

270-Mycobacterium

neoaurum VKM

Ac-1815D

194-Austwickia chelonae NBRC 105200

106-

Stre

ptom

yces

xia

men

ensi

s

4N

GB mudifib

muiretcabodifiB-703

424-Clostridium sp.CAG:628

79-Actinobacteria bacterium O

V450

266-Mycobacterium sm

egmatis.st.ATCC.700084

258-Mycobacterium leprae.st.TN

292-Bifidobacterium asteroides.st.PR

L2011

196-Curtobacterium flaccumfaciens UCD-AKU

384-Haloarcula marismortui.st.ATCC.43049

175-Corynebacterium sp.ATCC.6931

324-

Aero

mic

robi

um m

arin

um.D

SM.1

5272

7-miscellaneous Crenarchaeota group archaeon SMTZ-80

112-

Stre

ptom

yces

sp.

st.S

PB07

4

9-Burkholderiales bacterium GJ-E10

226-Arthrobacter sp.PAMC25486

66-Actinosynnema m

irum.st.ATCC.29888

131-

Stre

ptom

yces

zinc

iresis

tens

K42

3075

1.C

CTA.t

s.sit

necs

elod

a mu

iret

cabo

difiB

-503

159-Corynebacte

rium epidermidicanis

243-Rhodococcus jostii.st.RHA1

117-

Stre

ptom

yces

sp.

MM

G15

33

284-Mobiluncus curtisii.st.ATCC.43063

124-

Stre

ptom

yces

plu

ripot

ens

379-Halalkalicoccus jeotgali.st.DSM 18796

162-Corynebacterium efficiens.st.DSM 44549

181-Corynebacterium lipophiloflavum.DSM.44291

333-

Micr

omon

ospo

ra s

p.NR

RL B

-168

02

387-Haloquadratum walsbyi.st.DSM 16790

153-Corynebacte

rium va

riabile.st.

DSM 44702

360-M

ethan

otherm

obac

ter th

ermau

totrop

hicus

.st.ATCC.29

096

161-Corynebacte

rium glutamicum.st.

ATCC.13032

346-

Segn

ilipar

us ro

tundu

s.st.A

TCC.

BAA-

972

390-Natronobacterium gregoryi.st.ATCC.43098

278-Saccharomonospora glauca K62

225-Arthrobacter arilaitensis.st.DSM 16368

39-Vibrio campbellii.st.HY01

173-Corynebacterium aurimucosum.st.ATCC.700975

85-Streptomyces albus.st.ATC

C.21838

211-Beutenbergia cavernae.st.ATCC.BAA-8

70-Nocardiopsis alba.st.ATCC.BAA-216565-Nocardia sp.NRRL S-836


62-Saccharothrix sp.NRRL B-16348

275-Mycobacterium

abscessus.st.ATCC.19977

313-

Actin

omyc

es s

p.or

al ta

xon

848

str.F

0332

52-Frankia sp.st.EAN1pec

416-Parcubacteria

290-Actinomyces sp.oral taxon 175 str.F0384

25853.C

CTA.ts.ayelttac secymotpertS-79

160-Corynebacte

rium sp.KPL1989


99-S

trept

omyc

es v

iola

ceus

nige

r Tu

4113

58-Stackebrandtia nassauensis.st.DSM 44728

250-Gordonia bronchialis.st.ATCC.25592

274-Mycobacterium

thermoresistibile ATCC.19527

216-Microbacterium sp.Ag1

388-Halogeometricum borinquense.st.ATCC.700274

329-

Salin

ispor

a tro

pica

.st.A

TCC.

BAA-

916

68-Thermobispora bispora.st.ATCC.19993

46-Deinococcus maricopensis.st.DSM 21211

369-Thermofilu

m pendens.st.H

rk 5

102-

Stre

ptom

yces

rube

llom

urin

us

215-Microbacterium sp.SA39406-Pyrococcus abyssi.st.GE5

34-Caldivirga maquilingensis.st.ATCC.700844

154-Corynebacte

rium jeike

ium.st.K411

47-Deinococcus soli.Cha et al.2014

206-Xylanimonas cellulosilytica.st.DSM 15894

197-Leucobacter sp.Ag1

132-

Stre

ptom

yces

gris

eofla

vus T

u400

0

233-Arthrobacter aurescens.st.TC131

9-Ku

tzne

ria a

lbid

a.D

SM.4

3870

247-Gordonia sp.KTR9

107-

Stre

ptom

yces

sp.

NR

RL

F-66

02

402-Thermococcus kodakarensis.st.ATCC.BAA-918

182-Corynebacterium ureicelerivorans

73-Streptomyces katrae

257-Mycobacterium parascrofulaceum ATCC.BAA-614

17-Microgenomates

27-Lyngbya sp.st.PCC 8106

64-Lechevalieria aerocolonigenes

204-Jonesia denitrificans.st.ATCC.14870

244-Rhodococcus erythropolis.st.PR4

322-

Del

eted

-I4F2

P8265-Mycobacterium sp.VKM Ac-1817D 341-

Patu

libac

ter m

edica

men

tivor

ans

51-Frankia sp.EUN1f

126-

Stre

ptom

yces

inca

rnat

us

172-Corynebacterium casei LMG S-19264

185-Streptosporangium roseum.st.ATCC.12428


80-Streptomyces sp.W

M6372

376-Natronobacterium gregoryi.st.ATCC.43098

396-Natrialba magadii.st.ATCC.43099

43-Deinococcus gobiensis.st.DSM 21396

281-Actinomyces graevenitzii C83

423-Trichoplax adhaerens36-Methanocella paludicola.st.DSM 17711

373-Candidatus Magnetobacte

rium bavaricum

30-Thermoproteus sp.AZ2

22-Hirschia baltica.st.ATCC.4981429-Jeotgalibacillus sp.D5

256-Mycobacterium paratuberculosis.st.ATCC.BAA-968

212-Microbacterium testaceum.st.StLB037

293-Bifidobacterium pseudolongum

PV8-2

50-Frankia symbiont subsp.Datisca glomerata

285-Mobiluncus m

ulieris ATCC.35239

163-Corynebacterium kutscheri

44-Deinococcus gobiensis.st.DSM 21396

38-Paraglaciecola arctica BSs20135

114-

Actin

obac

teria

bac

teriu

m O

K006

109-

Stre

ptom

yces

var

iega

tus

418-Pelagibacter ubique.st.HTCC1062

378-Halobacterium salinarum.st.ATCC.700922

53-Frankia sp.st.EuI1c

151-C

oryne

bacte

rium fa

lsenii.

DSM.4435

3

32-Ignisphaera aggregans.st.DSM 17230

37-Paraglaciecola arctica BSs20135

49-Frankia alni.st.ACN14a

143-S

trepto

myces

caele

stis

404-Thermococcus barophilus.st.DSM 11836

277-Nocardia nova SH22a

320-

Geo

derm

atop

hilu

s ob

scur

us.s

t.ATC

C.2

5078

395-Haloterrigena turkmenica.st.ATCC.51198

168-Corynebacterium maris.DSM.45190

125-

Stre

ptom

yces

col

linus

.st.D

SM 4

0733

391-Halopiger xanaduensis.st.DSM 18323

232-Arthrobacter sp.st.FB24

411-Metagenomes

296-Scardovia inopinata F0304

400-Crenarchaeota group-1 archaeon SG8-32-1

11-Synechococcus sp.ATCC.27264

214-Microbacterium hydrocarbonoxydans

230-Arthrobacter globiformis NBRC 12137

195-Kineococcus radiotolerans.st.ATCC.BAA-149

19-Chryseobacterium sp.ERMR1:04

86-Streptomyces sp.PVA 94-07

370-Lokiarch

aeum sp.GC14 75

128-

Stre

ptom

yces

rose

ochr

omog

enus

subs

p.os

citan

s DS

12.9

76

189-Terrabacter sp.28

8900

2.MS

D.mu

talu

gna

muir

etca

bodif

iB-6

03

399-Crenarchaeota group-6 archaeon AD8-1

299-Bifidobacterium gallicum

.DSM

.20093

75-Streptomyces sp.M

g1

377-Halobacterium sp.DL1

72-Nocardiopsis sp.NRRL B-16309

136-

Stre

ptomyc

es gl

auce

scen

s

205-Cellulomonas gilvus.st.ATCC.13127

84-Streptomyces venezuelae.st.ATC

C.10712

291-Catenulispora acidiphila.st.D

SM 44928

380-Natronomonas moolapensis.st.DSM 18674

5-Methanococcoides burtonii.st.DSM 6242

15-Streptomyces pluripotens

202-Rothia dentocariosa.st.ATCC.17931

78-Streptomyces sp.N

RR

L F-4428

342-

Cone

xibac

ter w

oese

i.st.D

SM 1

4684

340-

Prop

ionim

icrob

ium ly

mph

ophil

um A

CS-0

93-V

-SCH

5

148-C

oryne

bacte

rium di

phthe

riae.s

t.ATCC.70

0971

321-

Blas

toco

ccus

sax

obsi

dens

.st.D

D2

122-

Stre

ptom

yces

sp.

PBH5

3

146-B

reviba

cteriu

m mcb

rellne

ri ATCC.49

030

4-Methanococcoides methylutens MM1

386-Haloferax volcanii.st.ATCC.29605

190-Intrasporangium calvum.st.ATCC.23552

45-Deinococcus proteolyticus.st.ATCC.35074

179-Corynebacterium marinum.DSM.44953

100-

Stre

ptom

yces

him

asta

tinic

us A

TCC

.536

53

245-Rhodococcus pyridinivorans SB3094

334-

Actin

opla

nes

friul

iens

is.DS

M.7

358

231-Renibacterium salmoninarum.st.ATCC.33209

219-Microbacterium ketosireducens


349-

Aero

pyru

m pern

ix.st.

ATCC.70

0893

273-Mycobacterium

hassiacum.DSM

.44199

261-Mycobacterium marinum.st.ATCC.BAA-535

405-Thermococcus litoralis.st.ATCC.51850

283-Actinomyces neuii BVS029A5

108-

Stre

ptom

yces

vie

tnam

ensi

s

223-Microbacterium chocolatum

135-

Stre

ptomyc

es da

vawe

nsis

JCM 49

13

359-M

ethan

obac

terium

sp.M

B1

133-

Stre

ptom

yces

sp.e

14

91-Streptomyces clavuligerus.st.ATC

C.27064

372-Candidatus Magnetobacte

rium bavaricum

279-Saccharomonospora m

arina XMU15

20A sucidyl secymotpertS-69

63-Saccharothrix espanaensis.st.ATCC.51144

152-Corynebacte

rium glyc

iniphilum AJ 3170

123-

Stre

ptom

yces

hyg

rosc

opicu

s su

bsp.

jingg

ange

nsis.

st.5

008

410-Metagenomes

298-Bifidobacterium therm

ophilum R

BL67

71-Nocardiopsis dassonvillei.st.ATCC.23218

414-Peptococcaceae bacterium SCADC1 2 3

16-Kitasatospora setae.st.ATCC.33774

171-Corynebacterium striatum ATCC.6940

167-Corynebacterium genitalium ATCC.33030

352-T

herm

oglad

ius ce

llulol

yticu

s.st.1

633 371-Lokia

rchaeum sp

.GC14 75

201-Rothia mucilaginosa.st.DY-18

25-Methanobacterium lacus.st.AL-21

220-Microbacterium trichothecenolyticum

210-Sanguibacter keddieii.st.ATCC.51767

260-Mycobacterium tuberculosis.st.ATCC.25618

6-Methanosalsum zhilinae.st.DSM 4017

164-Corynebacterium mustelae

69-Thermom

onospora curvata.st.ATCC.19995

139-

Stre

ptomyc

es sp

.NRRL W

C-361

8

89-Streptomyces sp.st.SirexAA-E

121-

Stre

ptom

yces

sp.

MUS

C119

T

365-Methanosphaera sta

dtmanae.st.

ATCC.43021

203-Kocuria rhizophila.st.ATCC.9341

295-Parascardovia denticolens.DSM

.10105

417-Brachyspira sp.CAG:700

14-Streptomyces albus.st.ATCC.21838

199-Clavibacter michiganensis.st.ATCC.33113

343-

Ilum

atob

acte

r coc

cineu

s YM

16-3

04

409-Bacillus cereus.st.ATCC.14579

156-Corynebacte

rium glucuronolyticum ATCC.51866

227-Arthrobacter chlorophenolicus.st.ATCC.700700

316-

Amyc

olat

opsi

s or

ient

alis

HC

CB1

0007

140-S

trepto

myces

virid

ochro

mogen

es

183-Corynebacterium testudinoris

103-

Sacc

haro

thrix

sp.

ST-8

88

145-S

trepto

myces

sp.W

M6378

149-C

oryne

bacte

rium re

sisten

s.st.D

SM 4510

0

355-H

ypert

herm

us bu

tylicu

s.st.D

SM 5456

55-Nocardioides sp.CF8

332-

Micr

omon

ospo

ra lu

pini

str.

Lupa

c 08

276-Dietzia cinnamea P4

10-Desulfatitalea sp.BRH c12

253-Nocardia farcinica.st.IFM 10152

208-Cellulomonas flavigena.st.ATCC.482

310-

Bifid

obac

teriu

m lo

ngum

.st.N

CC

270

5

67-Thermobifida fusca.st.YX

325-

Actin

opla

nes

miss

ourie

nsis.

st.A

TCC.

1453

8

269-Deleted- L0J2N8

56-Nocardioides sp.st.BAA-499

401-Candidatus Caldiarchaeum subterraneum

394-Natronococcus occultus SP4

353-T

herm

osph

aera

aggre

gans

.st.D

SM 1148

6

224-Kocuria sp.UCD-OTCP

158-Corynebacte

rium argentoratense.DSM.44202

315-

Stre

ptom

yces

sp.

AA4

218-Microbacterium foliorum

967.ps secymotpertS-59

354-a

rchae

on G

W20

11 AR10

222-Microbacterium laevaniformans OR221

20-Citreicella sp.357

347-

Acido

therm

us ce

llulol

yticu

s.st.A

TCC.

4306

8

57-Nocardioides luteus

2-Methanomethylovorans hollandica.st.DSM 15978

362-M

ethan

obrev

ibacte

r sp.A

bM4

141-S

trepto

myces

sp.A

S58

59-Nakamurella m

ultipartita.st.ATCC.700099

74-Streptomyces sp.C

48-Frankia sp.st.CcI3

213-Microbacterium azadirachtae

118-

Stre

ptom

yces

sp.

MM

G11

21

129-

Stre

ptom

yces

nod

osus

193-Mobilicoccus pelagius NBRC 104925

82-Streptomyces niveus N

CIM

B 11891

138-

Stre

ptomyc

es sc

abiei

.st.87

.22

344-

Micr

oluna

tus p

hosp

hovo

rus.s

t.ATC

C.70

0054

358-A

cidian

us ho

spita

lis.st.

W1

142-S

trepto

myces

ghan

aens

is AT

CC.1467

2

367-Methanopyru

s kandleri.s

t.AV19

1000

RGA

sut

arua

sec

ymo

tpert

S-49

12-Desulfobacterium autotrophicum.st.ATCC.43914

229-Brachybacterium sp.SW0106-09


RR

L F-6602

236-Tsukamurella paurometabola.st.ATCC.8368

188-Janibacter sp.HTCC2649

302-Gardnerella vaginalis JC

P8481B

209-Cellulomonas fimi.st.ATCC.484

115-

Stre

ptom

yces

sp.

WM

6386

113-

Stre

ptom

yces

sp.

Tu60

71

178-Corynebacterium humireducens NBRC 106098

144-S

trepto

myces

cyan

eogri

seus

subs

p.non

cyan

ogen

us

176-Corynebacterium doosanense CAU 212

177-Corynebacterium halotolerans YIM 70093

104-

Stre

ptom

yces

sp.

NR

RL

S-49

5

286-Actinomyces turicensis ACS-279-V-Col4

267-Mycobacterium obuense

241-Rhodococcus sp.B7740

33-Vulcanisaeta distributa.st.DSM 14429

364-Methanobacte

rium paludis.s

t.DSM 25820

312-

Arca

noba

cter

ium

hae

mol

ytic

um.s

t.ATC

C.9

345

200-Leifsonia xyli subsp.xyli.st.CTCB07

8-Desulfurispirillum indicum.st.ATCC.BAA-1389

336-

Prop

ioni

bact

eriu

m p

ropi

onicu

m.s

t.F02

30a

363-M

ethan

obrev

ibacte

r smith

ii.st.P

S

166-Corynebacterium pseudotuberculosis.st.C231

259-Mycobacterium kansasii ATCC.12478


13-Streptomyces viridochromogenes

381-Halobacterium sp.DL1

403-Thermococcus gammatolerans.st.DSM 15229

184-Corynebacterium uterequi

330-

Micr

omon

ospo

ra s

p.HK

10

110-

Stre

ptom

yces

viri

doch

rom

ogen

es.s

t.DSM

407

36

76-Streptomyces sp.W

M4235

60-Saccharopolyspora erythraea.st.ATCC.11635

328-

Micr

omon

ospo

ra a

uran

tiaca

.st.A

TCC.

2702

9

242-Rhodococcus aetherivorans

407-Pyrococcus furiosus.st.ATCC.43587


RR

L F-6491

350-

Pyro

lobus

fumar

ii.st.D

SM 11

204

385-halophilic archaeon DL31

3-Methanolobus psychrophilus R15

170-Corynebacterium sp.KPL1855

77-Streptomyces sp.NRRL S-44412

7-St

rept

omyc

es g

riseo

aura

ntiac

us M

045

297-Scardovia wiggsiae F0424

301-Gardnerella vaginalis.st.ATC

C.14019

413-Rhodospirillaceae bacterium BRH c57

368-Thermofilu

m sp.1910b

268-Mycobacterium

vanbaalenii.st.DSM 7251

398-Halomicrobium mukohataei.st.ATCC.700874

422-Ricinus communis

186-Kytococcus sedentarius.st.ATCC.14392

314-

Actin

otig

num

sch

aalii

240-Rhodococcus equi ATCC.33707

147-C

oryne

bacte

rium m

atruc

hotii A

TCC.1426

6

40-Firmicutes bacterium CAG:114

8F mugnol.psbus

mugnol muiretcabodifiB-903

389-Halorubrum lacusprofundi.st.ATCC.4923910

5-St

rept

omyc

es s

p.C

NQ

-509

234-Micrococcus luteus.st.ATCC.4698

361-M

ethan

obrev

ibacte

r rumina

ntium

.st.ATCC.35

063

130-

Stre

ptom

yces

coeli

color

.st.A

TCC.

BAA-

471

335-

Actin

opla

nes

sp.A

TCC.

3104

4

263-Mycobacterium sp.EPa45

251-Gordonia rhizosphera NBRC 16068

235-marine actinobacterium PHSC20C1

338-

Prop

ioniba

cteriu

m a

cnes

.st.K

PA17

1202

318-

Sacc

haro

mon

ospo

ra v

iridi

s.st

.ATC

C.1

5386

150-C

oryne

bacte

rium ur

ealyti

cum.st

.ATCC.4304

2

382-Halobacterium salinarum.st.ATCC.700922

157-Corynebacte

rium atypicu

m

198-Frigoribacterium sp.RIT-PI-h

24-Burkholderia sp.YI23

116-

Stre

ptom

yces

ave

rmitil

is.st

.ATC

C.31

267

272-Mycobacterium

rhodesiae.st.NBB3

119-

Stre

ptom

yces

sp.

MUS

C136

T

246-Nocardia brasiliensis ATCC.700358

397-Halostagnicola larsenii XH-48

248-Gordonia amarae NBRC 15530

303-Bifidobacterium scardovii

374-Candidatus Magnetobacterium bavaricum

134-

Stre

ptomyc

es m

angr

oviso

li

252-Nocardia cyriacigeorgica.st.GUH-2

81-Streptomyces tsukubensis N

RR

L18488

331-

Verru

cosis

pora

mar

is.st

.AB-

18-0

32

392-Natrinema sp.st.J7-2

357-S

ulfolo

bus s

olfata

ricus

.st.AT

CC.3509

2

18-Amphimedon queenslandica

383-Natronomonas pharaonis.st.ATCC.35678

280-Actinomyces m

assiliensis F0489

300-Bifidobacterium dentium

.st.ATCC

.27534

207-Isoptericola variabilis.st.225

327-

Actin

opla

nes

sp.N

902-

109

191-Janibacter hoylei PVAS-1

239-Gordonia sputi.NBRC.100414

326-

Pseu

dono

card

ia d

ioxa

nivo

rans

.st.A

TCC.

5548

6

255-Mycobacterium xenopi RIVM700367

264-Mycobacterium sinense.st.JDM601

421-Amphimedon queenslandica

21-Brevundimonas sp.AAP58

90-Streptomyces pratensis.st.ATC

C.33331

26-Lyngbya sp.st.PCC 8106

408-Methanocaldococcus jannaschii.st.ATCC.43067

238-Gordonia polyisoprenivorans.st.DSM 44266

393-Natrinema pellirubrum.st.DSM 15624

412-Thiothrix nivea.DSM.5205

187-Dermacoccus nishinomiyaensis

31-Pyrobaculum aerophilum.st.ATCC.51768

311-

True

pere

lla p

yoge

nes

155-Corynebacte

rium kroppenste

dtii.st.D

SM 44385

356-S

ulfolo

bus t

okod

aii.st

.DSM 16

993

180-Corynebacterium imitans

317-

Amyc

olat

opsi

s m

edite

rrane

i.st.S

699

323-

Krib

bella

flav

ida.

st.D

SM 1

7836

221-Microbacterium ginsengisoli

42-Deinococcus swuensis

254-Mycobacterium elephantis

174-Corynebacterium xerosis

137-

Actin

obac

teria

bacte

rium O

K074

165-Corynebacterium vitaeruminis.DSM.20294

351-

Stap

hylot

herm

us m

arinu

s.st.A

TCC.43

588

120-

Stre

ptom

yces

svic

eus A

TCC.

2908

3

6845

2.C

CTA

silari

psea

nitsir

p se

cymo

tpert

S-29

101-

Kita

sato

spor

a se

tae.

st.A

TCC

.337

74

366-Methanobacte

rium lacus.s

t.AL-21

110

DA.t

s.sit

cal.

psbu

s sil

ami

na

muir

etca

bodif

iB-4

03

289-Actinomyces urogenitalis.DSM

.15434

271-Mycobacterium

sp.st.KMS

88-Streptomyces griseus subsp.griseus.st.JC

M 4626

1-Firmicutes bacterium CAG:582

337-

Prop

ioni

bact

eriu

m a

cidip

ropi

onici

.st.A

TCC.

4875

237-Rhodococcus sp.RD6.2

28-Bacillus decisifrondis

339-

Prop

freu

denr

eichii

subs

p.sh

erm

anii.s

t.ATC

C.96

14

192-Kineosphaera limosa NBRC 100340

41-Frankia sp.EUN1f

98-S

trept

omyc

es b

ingc

heng

gens

is.s

t.BC

W-1

61-Amycolicicoccus subflavus.st.DSM

45089

217-Microbacterium oxydans

54-Nocardioides simplex

294-Bifidobacterium kashiw

anohense PV20-2

262-Mycobacterium haemophilum

345-

Actin

obac

teria

bac

teriu

m IM

CC26

256

375-Rhizophagus irregularis DAOM 197198w

228-Brachybacterium faecium.st.ATCC.43885

23-Burkholderia vietnamiensis.st.G4

Tree scale

9

Supplementary Figure 7. Phylogenetic analyses of NucS N-terminal (NT) region. Unrooted ML

tree of NT sequences from the PFAM PF01939 domain. Black branches, Actinobacteria; red

Deinococcus-Thermus; light blue, Archaea. Labels indicate species name. The circles represent

>80% bootstrap in 1,500 replicates.

115-Bifidobacterium_breve.DSM.20213

34-Streptomyces_albus.st.ATCC.21838

166-Natronomonas_pharaonis.st.ATCC.35678

97-Amycolicicoccus_subflavus.st.DSM_45089

289-Gordonia_amarae.NBRC.15530

254-Curtobacterium

_flaccumfaciens_U

CD-AKU

273-Mycobacterium_hassiacum.DSM.44199

108-Bifidobacterium_bifidum_BGN4

42-Streptomyces_sp.NRRL_S-495

84-Amycolatopsis_orientalis_HCCB10007

262-

325-Cory

nebacteri

um_vitae

ruminis.D

SM.20294

6-Streptomyces_lydicus_A02

131-Halomicrobium_mukohataei.st.ATCC.700874

308-Corynebacterium_humireducens.NBRC.106098_=.DSM.45392

159-Methanobrevibacter_smithii.st.PS

247-Arthrobacter_sp.PAMC25486

120-Actinomyces_sp.oral_taxon_848_str.F0332

285-Gordonia_sputi.NBRC.100414

126-halophilic_archaeon_DL31

161-Methanosphaera_stadtmanae.st.ATCC.43021

179-Halogeometricum_borinquense.st.ATCC.700274

12-Streptomyces_sp.NRRL_WC-3618

337-Corynebacter

ium_jeikeium.st.K4

11

143-Natro

nobacteriu

m_gregor

yi.st.ATCC

.43098

344-Propionibacterium_acidipropionici.st.ATCC

.4875

151-Pyrococcu

s_abyssi.st.GE

5

93-Nocardia_nova_SH22a

216-Microbacterium_ketosireducens

354-Thermobifida_fusca.st.YX

279-Mycobacterium_bovis

226-Cellulomonas_gilvus.st.ATCC.13127

40-Streptomyces_caelestis

358-Frankia_sp.st.EuI1c


170-Halomicrobium_mukohataei.st.ATCC.700874

43-Streptomyces_rubellom

urinus

76-Streptomyces_zinciresistens_K42

1-Streptomyces_bingchenggensis.st.BCW-1

299-Corynebacterium_xerosis

275-Mycobacterium_marinum.st.ATCC.BAA-535

315-Corynebacterium

_aurimucosum

.st.ATC

C.700975

51-Actinoplanes_missouriensis.st.ATCC.14538

78-Pseudonocardia_dioxanivorans.st.ATCC.55486

293-Rhodococcus_sp.B7740

101-Brevibacterium_mcbrellneri.ATCC.49030

230-Isoptericola_variabilis.st.225

21-Actinobacteria_bacterium_OK006

74-Actinobacteria_bacterium_OV320

242-Brachybacterium_faecium

.st.ATCC.43885

133-Halopiger_xanaduensis.st.D

SM_18323

45-Verrucosispora_maris.st.AB-18-032

105-Gardnerella_vaginalis_JCP8481B

220-Microbacterium_sp.SA39

110-Bifidobacterium_pseudolongum_PV8-2

272-Mycobacterium_neoaurum_VKM_Ac-1815D

227-Cellulomonas_flavigena.st.ATCC.482

152-Methanocal

dococcus_janna

schii.st.ATCC.43

067

33-Streptomyces_glaucescens

46-Microm

onospora_sp.HK10

212-Microbacterium_laevaniformans_OR221

165-Methanobacterium_sp.MB1

66-Streptomyces_sp.PBH

53

198-Methanocella_paludicola.st.DSM_17711

318-Coryne

bacteriu

m_glutamicum.st.ATCC

.13032

319-Coryne

bacteriu

m_argen

toratens

e.DSM.442

02

180-Haloferax_volcanii.st.ATCC.29605

326-Cory

nebacteriu

m_kutsche

ri

79-Saccharopolyspora_erythraea.st.ATCC.11635

13-Streptomyces_avermitilis.st.ATCC.31267

69-Streptomyces_pratensis.st.ATC

C.33331


122-Halobacterium_salinarum.st.ATCC.700922

7-Streptomyces_auratus_AGR0001

50-Actinoplanes_sp.st.ATCC.31044304-Turicella_otitidis.ATCC.51513

39-Streptomyces_griseoflavus_Tu4000

113-Bifidobacterium_kashiwanohense_PV20-2

169-Haloarcula_marismortui.st.ATCC.43049

184-Caldivirga_maquilingensis.st.ATCC.700844

144-Haloba

cterium_sa

linarum.st.

ATCC.7009

22

47-Microm

onospora_sp.NRRL_B-16802

11-Streptomyces_sp.AS58

250-Arthrobacter_chlorophenolicus.st.ATCC.700700

67-Streptomyces_hygroscopicus_subsp.jinggangensis.st.5008

53-Microm

onospora_lupini_str.Lupac_08

27-Streptomyces_sp.C

17-Streptomyces_tsukubensis_NRRL18488

96-Stackebrandtia_nassauensis.st.DSM_44728

200-Conexibacter_woesei.st.DSM_14684

215-Microbacterium_trichothecenolyticum

291-Rhodococcus_aetherivorans

162-Methanothermobacter_thermautotrophicus.st.ATCC.29096

44-Saccharothrix_sp.ST-888

360-Frankia_symbiont_subsp.Datisca_glomerata

38-Streptomyces_griseus_subsp.griseus.st.JCM_4626

119-Rothia_mucilaginosa.st.DY-18

132-Halovivax_ruber.st.D

SM_18193

248-Arthrobacter_globiformis.N

BRC.12137

61-Streptomyces_m

angrovisoli

52-Actinoplanes_friuliensis.DSM.7358

62-Streptomyces_cyaneogriseus_subsp.noncyanogenus

98-Kutzneria_albida.DSM.43870

30-Streptomyces_sp.Mg1

37-Streptomyces_vietnamensis48-M

icromonospora_aurantiaca.st.ATCC.27029

300-Corynebacterium_sp.ATCC.6931

332-Coryneb

acterium_var

iabile.st.DSM

_44702

71-Streptomyces_sp.M

MG1121

362-Streptomyces_violaceusniger_Tu_4113

234-Austwickia_chelonae.NBRC.105200

172-Natrinema_pellirubrum.st.DSM_15624

218-Microbacterium_azadirachtae

178-halophilic_archaeon_DL31

194-Deinococcus_swuensis

14-Streptomyces_nodosus

245-Mobiluncus_curtisii.st.ATC

C.43063

25-Streptomyces_variegatus

100-Kytococcus_sedentarius.st.ATCC.14392

160-Methanobrevibacter_sp.AbM4

357-Frankia_sp.st.CcI3

127-Haloarcula_marism

ortui.st.ATCC.43049

134-Halostagnicola_la

rsenii_XH-48

189-Staphylothermus_marinus.st.ATCC.43588

309-Corynebacterium

_marinum.DSM.44953

139-The

rmofilum

_pendens

.st.Hrk_5

295-Rhodococcus_equi.ATCC.33707

5-Streptomyces_sp.769

298-Dietzia_cinnamea_P4

345-Propionimicrobium_lymphophilum_ACS-093-V-SCH5

322-Cor

ynebacte

rium_pse

udotube

rculosis.s

t.C231

258-Leifsonia_xyli_subsp.xyli.st.CTCB07

4-Streptomyces_rimosus_subsp.rimosus.ATCC.10970

111-Bifidobacterium_dentium.st.ATCC.27534

123-Halorubrum_lacusprofundi.st.ATCC.49239

190-Hyperthermus_butylicus.st.DSM_5456

241-Brachybacterium_sp.SW

0106-09

361-Frankia_sp.st.EAN1pec

103-Nocardioides_luteus

36-Streptomyces_sp.NRRL_F-6491

228-Jonesia_denitrificans.st.ATCC.14870

367-Streptomyces_sp.Tu6071

181-Haloquadratum_walsbyi.st.DSM_16790338-Corynebacteri

um_lipophiloflavum.

DSM.44291

233-Kineosphaera_limosa.NBRC.100340

114-Bifidobacterium_animalis_subsp.lactis.st.AD011

60-Streptomyces_sp.e14

321-Coryne

bacteriu

m_diphtheriae.s

t.ATCC.

700971

333-Coryneba

cterium_glycin

iphilum_AJ_3

170

70-Streptomyces_incarnatus

349-Nocardiopsis_sp.NRRL_B-16309

264-Mycobacterium_sp.st.KMS

288-Gordonia_rhizosphera.NBRC.16068

252-Arthrobacter_arilaitensis.st.DSM_16368

83-Amycolatopsis_mediterranei.st.S699

195-Deinococcus_gobiensis.st.DSM_21396

31-Streptomyces_sp.WM6372

290-Gordonia_sihwensis.NBRC.108236

137-Haloqu

adratum_wa

lsbyi.st.D

SM_16790

278-Mycobacterium_tuberculosis.st.ATCC.25618

94-Nocardia_brasiliensis.ATCC.700358

281-Mycobacterium_haemophilum

327-Cory

nebacteriu

m_halotole

rans_YIM_

70093

282-Mycobacterium_paratuberculosis.st.ATCC.BAA-968

294-Rhodococcus_erythropolis.st.PR4

121-Actinomyces_neuii_BVS029A5

125-Haloferax_volcanii.st.ATCC.29605

77-Streptomyces_cattleya.st.ATCC.35852

246-Kocuria_sp.UCD-OTCP

155-Thermococcus_b

arophilus.st.DSM_118

36

206-Actinotignum_schaalii



55-Catenulispora_acidiphila.st.DSM_44928

259-Streptomyces_sp.AA4

263-Mycobacterium_elephantis

138-The

rmofilum

_sp.191

0b

91-Nocardia_farcinica.st.IFM_10152

168-Halalkalicoccus_jeotgali.st.DSM_18796

146-Methan

ocella_palu

dicola.st.DS

M_17711

204-Actinomyces_massiliensis_F0489

302-Corynebacterium_ureicelerivorans

323-Cory

nebacteri

um_matr

uchotii.AT

CC.1426

6

297-Rhodococcus_pyridinivorans_SB3094


346-Propionibacterium_propionicum.st.F0230a

148-Acidian

us_hospitalis

.st.W1

320-Coryne

bacteriu

m_efficie

ns.st.DS

M_44549

154-Thermococcus_

gammatolerans.st.D

SM_15229

92-Nocardia_cyriacigeorgica.st.GUH-2

229-Cellulomonas_fim

i.st.ATCC.484140-

miscellan

eous_Cre

narchaeo

ta_group

-6_archa

eon_AD8

-1

54-Salinispora_tropica.st.ATCC.BAA-916

49-Actinoplanes_sp.N902-109

185-Thermoproteus_sp.AZ2

188-Thermosphaera_aggregans.st.DSM_11486

106-Bifidobacterium_thermophilum_RBL67

329-Coryne

bacterium_

sp.KPL198

9

142-Halo

bacterium

_sp.DL1

28-Streptomyces_katrae

102-Aeromicrobium_marinum.DSM.15272

219-Microbacterium_sp.Ag1

336-Corynebact

erium_resistens.s

t.DSM_45100

210-Arcanobacterium_haemolyticum.st.ATCC.9345

277-Mycobacterium_parascrofulaceum.ATCC.BAA-614

213-Microbacterium_testaceum.st.StLB037

312-Corynebacterium

_casei_LM

G_S-19264

117-Nocardioides_sp.st.BAA-499

348-Nocardiopsis_dassonvillei.st.ATCC.23218

328-Coryne

bacterium_

testudinor

is

271-Mycobacterium_smegmatis.st.ATCC.700084

239-Kineococcus_radiotolerans.st.ATCC.BAA-149

58-Streptomyces_niveus_N

CIMB_11891

301-Corynebacterium_epidermidicanis

72-Streptomyces_sp.M

USC136T

368-Streptomyces_clavuligerus.st.ATCC.27064

355-Acidothermus_cellulolyticus.st.ATCC.43068

225-Xylanimonas_cellulosilytica.st.DSM

_15894

158-Methanobrevibacter_rumin

antium.st.ATCC.35063

317-Corynebacterium_uterequi

211-Trueperella_pyogenes

221-Microbacterium_hydrocarbonoxydans

99-Nakamurella_multipartita.st.ATCC.700099

356-Frankia_alni.st.ACN14a

196-Deinococcus_proteolyticus.st.ATCC.35074

116-Nocardioides_sp.CF8

59-Streptomyces_pluripotens

2-Streptomyces_sp.CNQ-509

141-misc

ellaneous

_Crenarc

haeota_g

roup-1_a

rchaeon_

SG8-32-1

81-Saccharomonospora_marina_XMU15

244-Kocuria_rhizophila.st.ATCC.9341

65-Streptomyces_roseochrom

ogenus_subsp.oscitans_DS_12.976

217-Microbacterium_ginsengisoli

269-Mycobacterium_vanbaalenii.st.DSM_7251

128-Natronomonas_m

oolapensis.st.DSM_18674

193-Marinithermus_hydrothermalis.st.DSM_14884

75-Streptomyces_viridochromogenes

347-Microlunatus_phosphovorus.st.ATCC.700054

145-Methan

ocella_arvo

ryzae.st.DS

M_22066

359-Frankia_sp.EUN1f

10-Streptomyces_griseoaurantiacus_M045

9-Streptomyces_pristinaespiralis.ATCC.25486

223-Microbacterium

_oxydans

237-Janibacter_hoylei_PVAS-1

306-Corynebacterium_glucuronolyticum.ATCC.51866

118-Rothia_dentocariosa.st.ATCC.17931

23-Streptomyces_sviceus.ATCC.29083

176-Natronococcus_occultus_SP4

56-Kitasatospora_setae.st.ATCC.33774

311-Corynebacterium

_doosanense_CAU_212_=.DSM.45436

296-Rhodococcus_jostii.st.RHA1

173-Natronobacterium_gregoryi.st.ATCC.43098

174-Natrinema_sp.st.J7-2

270-Mycobacterium_obuense

283-Mycobacterium_sp.EPa45

183-Vulcanisaeta_distributa.st.DSM_14429

32-Streptomyces_sp.NRRL_S-444

268-Mycobacterium_thermoresistibile.ATCC.19527

149-Sulfolobu

s_acidocalda

rius.st.ATCC.

33909

314-Corynebacterium

_camporealensis

8-Streptomyces_xiamenensis

340-

366-Streptomyces_sp.st.SPB074

164-Methanobacterium_paludis.st.DSM_25820

186-Pyrobaculum_aerophilum.st.ATCC.51768

257-Frigoribacterium_sp.RIT-PI-h

207-Actinomyces_turicensis_ACS-279-V-Col4

87-Saccharothrix_espanaensis.st.ATCC.51144


240-Beutenbergia_cavernae.st.ATCC.BAA-8

109-Bifidobacterium_gallicum.DSM.20093

286-Gordonia_bronchialis.st.ATCC.25592

182-Ignisphaera_aggregans.st.DSM_17230

197-Thermogladius_cellulolyticus.st.1633

334-Coryneba

cterium_urealy

ticum.st.ATCC

.43042

260-Tsukamurella_paurometabola.st.ATCC.8368

112-Bifidobacterium_adolescentis.st.ATCC.15703

80-Saccharomonospora_glauca_K62

64-Streptomyces_viridochrom

ogenes.st.DSM_40736

305-Corynebacterium_genitalium.ATCC.33030

15-Streptomyces_sp.MMG1533

86-Actinosynnema_mirum.st.ATCC.29888

324-Cory

nebacteri

um_mus

telae

351-Streptosporangium_roseum.st.ATCC.12428

287-Gordonia_sp.KTR9

167-Natronomonas_moolapensis.st.DSM_18674

82-Saccharomonospora_viridis.st.ATCC.15386

104-Nocardioides_simplex

187-Candidatus_Caldiarchaeum_subterraneum

316-Corynebacterium_striatum.ATCC

.6940

352-Thermobispora_bispora.st.ATCC.19993


280-Mycobacterium_leprae.st.TN

343-Propionibacterium_freudenreichi

i_subsp.shermanii.st.ATCC.9614

147-Sulfolob

us_solfatari

cus.st.ATCC

.35092

214-Microbacterium_chocolatum

156-Thermococcus_litor

alis.st.ATCC.51850

235-Mobilicoccus_pelagius.NBRC.104925

364-Streptomyces_sp.PVA_94-07

129-Natronomonas_pharaonis.st.ATCC.35678

171-Halostagnicola_larsenii_XH-48

339-Corynebacterium

_kroppenstedtii.st.DS

M_44385

267-Mycobacterium_xenopi_RIVM700367

255-Clavibacter_m

ichiganensis_subsp.sepedonicus.st.ATCC.33113

342-Geodermatophilus_obscuru

s.st.ATCC.25078

107-Bifidobacterium_angulatum.DSM.20098

153-Pyrococcus_f

uriosus.st.ATCC.4

3587

199-archaeon_GW2011_AR10

150-Sulfolobu

s_tokodaii.st.D

SM_16993

191-Deinococcus_maricopensis.st.DSM_21211

89-Lechevalieria_aerocolonigenes

157-Thermococcus_kodak

arensis.st.ATCC.BAA-918

276-Mycobacterium_kansasii.ATCC.12478

19-Actinobacteria_bacterium_OV450


249-Arthrobacter_sp.st.FB24


238-Janibacter_sp.HTCC2649

253-Leucobacter_sp.Ag1

307-Corynebacterium_atypicum

135-Natrialba_mag

adii.st.A

TCC.430

99

57-Streptomyces_davaw

ensis_JCM_4913

256-marine_actinobacterium

_PHSC20C

1

85-Nocardia_sp.NRRL_S-836

335-Corynebac

terium_falsenii.

DSM.44353

266-Mycobacterium_rhodesiae.st.NBB3

63-Streptomyces_collinus.st.D

SM_40733

35-Actinobacteria_bacterium_OK074

68-Streptomyces_sp.st.SirexAA-E

203-Actinomyces_urogenitalis.DSM.15434

350-Nocardiopsis_alba.st.ATCC.BAA-2165

284-Gordonia_polyisoprenivorans.st.DSM_44266

303-Corynebacterium_imitans

331-Ilumato

bacter_coc

cineus_YM1

6-304

330-Actinob

acteria_bac

terium_IMC

C26256

236-Dermacoccus_nishinom

iyaensis261-Mycobacterium_abscessus.st.ATCC.19977

16-Streptomyces_venezuelae.st.ATCC.10712

310-Corynebacterium

_maris.DSM.45190

243-Micrococcus_luteus.st.ATC

C.4698


313-Corynebacterium

_sp.KPL1855

224-Sanguibacter_keddieii.st.ATCC.51767

201-Actinomyces_graevenitzii_C83

163-Methanobacterium_lacus.st.AL-21

353-Thermomonospora_curvata.st.ATCC.19995

124-Halogeometricum_borinquense.st.ATCC.700274

95-Kribbella_flavida.st.DSM_17836

251-Renibacterium

_salmoninarum

.st.ATCC.33209

175-Halopiger_xanaduensis.st.DSM_18323

231-Terrabacter_sp.28

88-Saccharothrix_sp.NRRL_B-16348

24-Streptomyces_sp.MUSC119T

363-Streptomyces_himastatinicus.ATCC.53653

292-Rhodococcus_sp.RD6.2

130-Halorhabdus_tiam

atea_SARL4B

274-Mycobacterium_sp.VKM_Ac-1817D

90-Segniliparus_rotundus.st.ATCC.BAA-972

222-Microbacterium_foliorum

341-Blastococcus_saxobsi

dens.st.DD2

26-Streptomyces_ghanaensis.ATCC.14672

192-'Deinococcus_soli'_Cha_et_al._2014

41-Streptomyces_coelicolor.st.ATCC.BAA-471

136-Natron

obacterium_gre

goryi.st.ATC

C.43098

265-Mycobacterium_sinense.st.JDM601

177-Halorubrum_lacusprofundi.st.ATCC.49239

232-Intrasporangium_calvum

.st.ATCC.23552

18-Streptomyces_scabiei.st.87.22

Tree scale: 1

ACTINO

BACT

ERIA ARCHAEA

ARCHAEA

Deinococcus

Deinococcus

10

Supplementary Tables

Supplementary Table 1. Mutation rates of M. smegmatis and its ∆nucS derivatives. Rates of

spontaneous mutations conferring rifampicin (Rif-R) and streptomycin resistance (Str-R) of M.

smegmatis mc2 155 (WT), M. smegmatis ∆nucS and M. smegmatis ∆nucS complemented with the

wild-type nucS from M. smegmatis mc2 155 (nucSSm). Fold change indicates the increase in

mutation rate with respect to the strain M. smegmatis mc2 155 (set to 1). Mut Rate: mutation rate

(mutations per cell per generation).

Strain Genotype Mut Rate Rif 95% CI Fold Mut Rate

Str 95% CI Fold

mc2 155 WT 2.07x10-‐9 1.47-‐2.74x10-‐9 1 5.58x10-‐10 2.25-‐11.1x10-‐10 1

∆nucS ∆nucS 3.11x10-‐7 2.81-‐3.39x10-‐7 150.2 4.82x10-‐8 3.53-‐6.17x10-‐8 86.3

∆nucS/nucSSm Complemented 3.02x10-‐9 2.2-‐3.97x10-‐9 1.46 1.43x10-‐9 0.77-‐2.33x10-‐9 2.56

11

Supplementary Table 2. Mutational spectrum of M. smegmatis mc2 155 and its ∆nucS derivative.

A) Spontaneous mutations conferring rifampicin resistance found in 41 independent Rif-R mutants in

the WT and ∆nucS strains. The columns show the position of the mutations in the rpoB gene sequence,

the codon change (modified bases are in bold), the amino acid change caused by each mutation and the

number of independent Rif-R mutants isolated from mc2 155 (WT) or its ΔnucS derivative. B) Specificity

of base substitutions, summarized by class, produced in the WT and ∆nucS strains.

A)

Position

Codon change

Amino acid change

WT

ΔnucS

A 1295 G

GAC→GGC

Asp 432 Gly

0

1

C 1324 T

CAC→TAC

His 442 Tyr

6

6

C 1324 G

CAC→GAC

His 442 Asp

2

0

A 1325 G

CAC→CGC

His 442 Arg

13

20

A 1325 C

CAC→CCC

His 442 Pro

4

0

G 1334 A

CGT →CAT

Arg 445 His

0

6

G 1334 C

CGT→CCT

Arg 445 Pro

1

0

G 1334 T

CGT→CTT

Arg 445 Leu

2

0

C 1340 T

TCG→TTG

Ser 447 Leu

9

6

C 1340 G

TCG→TGG

Ser 447 Trp

2

0

T 1346 C

CTG→CCG

Leu 449 Pro

0

2

G 1348 A

GGC→AGC

Gly 450 Ser

1

0

Δ CCAGCTGTC (1275-1283)

CCAGCTGTC

Ser 425 Arg + ΔGlnLeuSer

(426-428)

1

0

TOTAL

41

41

12

B)

Mutation WT (%) n=41

∆nucS (%) n=41

G:C→A:T 16 (39.0) 18 (43.9) A:T→G:C 13 (31.7) 23 (56.1) G:C→T:A 2 (4.9) 0 A:T→T:A 0 0 G:C→C:G 5 (12.2) 0 A:T→C:G 4 (9.7) 0 Deletions 1 (2.4) 0

13

Supplementary Table 3. Mutation rates of S. coelicolor A3(2) M145 and its ∆nucS

derivatives. Rates of spontaneous mutations conferring rifampicin (Rif-R) and streptomycin

resistance (Str-R) of S. coelicolor A3(2) M145, its ∆nucS derivative and S. coelicolor ∆nucS

complemented with the wild-type nucS from S. coelicolor (nucSSco). 95% confidence intervals (CI)

are indicated. Mut Rate: mutation rate (mutations per cell per generation).

Strain Genotype Mut Rate Rif-R 95% CI Fold Mut Rate

Str-R 95% CI Fold

S. coelicolor

A3(2) M145 WT 5.11x10-9 2.87-8.01x10-9 1 4.30x10-10 0.24-18.9x10-10 1

∆nucS ∆nucS 5.54x10-7 4.20-6.97x10-7 108.4 8.49x10-8 5.78-11.4x10-8 197.4

∆nucS/nucSSco Complemented 2.01x10-9 0.83-3.88x10-9 0.5 4.30x10-10 0.24-18.9x10-10 1

14

Supplementary Table 4. Characteristics of the M. tuberculosis representative strains

containing NucS polymorphisms. Genome ID/common name, NucS polymorphism, resistance

profile, lineage and origin of the strains are shown. Resistance profile indicates not

detected/unknown antibiotic resistances (Susceptible) or MDR, multidrug-resistant strain

(expressing at least rifampicin and isoniazid resistance).

GenomeID/ name Polymorphism Resistance

profile Lineage Origin

CDC1551 WT Susceptible 4 North America TKK_02_0079 S39R MDR 4 South Africa MTB_N1057 S54I Susceptible 4 South Asia

KT-0040 A67S Susceptible 2 S. Korea (Broad Inst) ERR036236 V69A Susceptible 1 Unknown BTB 04-388 A135S MDR 3 Sweden (Broad Inst) BTB 07-246 R144S MDR 4 Sweden (Broad Inst)

TKK_03_0044 D162H Susceptible 4 South Africa HN2738 T168A Unknown Unknown Unknown (Broad Inst)

MTB_X632 K184E MDR 4 Central America

15

Supplementary Table 5. Effect of M. tuberculosis NucS naturally occurring polymorphisms on

mutation rates. Rates of spontaneous mutations conferring rifampicin resistance (Mut rate,

mutations per cell per generation) of M. smegmatis ∆nucS complemented with the wild-type nucS

from M. tuberculosis (nucSTB) or the 9 polymorphic alleles. 95% confidence intervals (CI) are

shown. Fold change indicates the increase in mutation rate with respect to the strain M. smegmatis

∆nucS complemented with wild-type nucSTB (∆nucS/nucSTB), set to 1.

Amino acid

change Codon change Mut rate 95% CI Fold change

M. smegmatis ∆nucS/nucSTB

nucSTB from CDC1551 4.17x10-9 3.29-5.12x10-9 1

S39R AGC→AGG 3.49x10-7 2.93-4.0x10-7 83.7

S54I AGT→ATT 7.94x10-9 5.49-11.07x10-9 1.9

A67S GCG→TCG 2.78x10-9 1.87-3.84x10-9 0.7

V69A GTG→GCG 8.77x10-9 6.50-11.2x10-9 2.1

A135S GCG→TCG 3.57x10-8 2.77-4.38x10-8 8.6

R144S CGC→AGC 3.16x10-8 2.40-4.0x10-8 7.6

D162H GAC→CAC 8.98x10-9 6.42-11.80x10-9 2.2

T168A ACC→GCC 3.96x10-8 3.09-4.81x10-8 9.5

K184E AAG→GAG 3.06x10-8 2.37-3.75x10-8 7.3

16

Supplementary Table 6. Sequence of oligonucleotides used in this work.

Oligonucleotide Sequence (5’-3’) Purpose

SecMycomar CCCGAAAAGTGCCACCTAAATTGTAAGCG Localization of Tn insertion site

DelnucSm5F CCCGCTGCAGCTGGCCGAGTTCGG nucSSm M. smegmatis deletion

DelnucSm5R CGGTAAGCTTGGCTATCACGAGGCGCACCC nucSSm M. smegmatis deletion

DelnucSm3F CGGAAAGCTTAGCGACGAGTACCGGCTCTT nucSSm M. smegmatis deletion

DelnucSm3R AATCGTCGACCGAACCCATCAACTTACCGA nucSSm M. smegmatis deletion

CompnucTBF ACTGGAATTCTCGAGTGGTGGCCTTCTCGGATGGCAT nucSTB for ΔnucSSm complementation

CompnucTBR ACTGAAGCTTTCAGAACAGCCGGTACTCGCCGCT nucSTB for ΔnucSSm complementation

CompnucSmF ACTGGAATTCCCGCGCCAGCGAATTGTCGGCGTTCAT nucSSm for ΔnucSSm complementation

CompnucSmR CGGCAAGCTTTCAGAAGAGCCGGTACTCGTCGCT nucSSm for ΔnucSSm complementation

RifRRDRrpoBF GTGGCGGCGATCAAGGAGTTCTTC RRDR-rpoBSm amplification

RifRRDRrpoBR GGCGACCGACACCATCTGGCGCGG RRDR-rpoBSm amplification

DelnucSco5F GTGTAAGCTTCGGCACCGCGGTGAGTGTGC nucSSco S. coelicolor deletion

DelnucSco5R CGACGGATCCGCGGGCAATGACGAGACGCA nucSSco S. coelicolor deletion

DelnucSco3F GCTGGGATCCTTCTGAGGGCGCGACGCGTC nucSSco S. coelicolor deletion

DelnucSco3R CAGGGATATCTGCCCGCCCTGGTCGGCGAG nucSSco S. coelicolor deletion

CompnucScoF TACGGAATTCGGGTTCTCCTCTCGCACCCCCGACCAGCAGGGG nucSSco for ΔnucSSco complementation

CompnucScoR TACGGGATCCTCAGAACAGCCGCAGCTTGTCGTCCTCGATGCC nucSSco for ΔnucSSco complementation

Rechyg3F CAGTTTCATTTGATGCTCGATGAG Recombination. pRhomyco 100%

Rechyg3R GACTAACTAGTCAGGCGCCGGGGGCGGTGT Recombination. pRhomyco 100%

Rechyg5F GATGCAGCTGGGAGTGCGCTGTGACACAAGAATCC Recombination. pRhomyco 100%

Rechyg5R CGATAAGCTTCGAATTCTGCAGCTCG Recombi. pRhomyco 100%, 95%, 90% and 85%

Rechyg5intR GATGTGATCACCGGGTCGGGCTCG Recombi. pRhomyco 95%, 90% and 85%

Rec95-BclIF GATGTGATCAAGCTGTTCGGGGAGCACTG Recombination. pRhomyco 95%

Rec95-NheIR GATGGCTAGCGAAGTCGACGATCCCGGTGA Recombination. pRhomyco 95%

Rec90-85-BclIF GATGTGATCAAGCTCTTCGGGGAACACTG Recombination. pRhomyco 90% and 85%

Rec90-85-PciIR GATGACATGTGAAGTCGACGATCCCGGTGA Recombination. pRhomyco 90% and 85%

S39RF GGCCGACGGATCGGTCAGGGTACATGCTGACGACCG Site-directed mutagenesis nucSTB

S39RR CGGTCGTCAGCATGTACCCTGACCGATCCGTCGGCC Site-directed mutagenesis nucSTB

S54IF CCGTTGAACTGGATGATTCCGCCGTGCTGGTTG Site-directed mutagenesis nucSTB

17

S54IR CAACCAGCACGGCGGAATCATCCAGTTCAACGG Site-directed mutagenesis nucSTB

A67SF AGAGTCCGGCGGCCAGTCGCCAGTGTGGGTGGTCG Site-directed mutagenesis nucSTB

A67SR CGACCACCCACACTGGCGACTGGCCGCCGGACTCTTC Site-directed mutagenesis nucSTB

V69AF CCGGCGGCCAGGCGCCAGCGTGGGTGGTCGAGAAC Site-directed mutagenesis nucSTB

V69AR GTTCTCGACCACCCACGCTGGCGCCTGGCCGCCGG Site-directed mutagenesis nucSTB

A135SF GCCGCGAGTACATGACCTCGATCGGACCCGTCGAC Site-directed mutagenesis nucSTB

A135SR GTCGACGGGTCCGATCGAGGTCATGTACTCGCGGC Site-directed mutagenesis nucSTB

A162HF GCGGCGTGGCGAGATCCACGGCGTGGAGCAGCTGAC Site-directed mutagenesis nucSTB

A162HR GTCAGCTGCTCCACGCCGTGGATCTCGCCACGCCGC Site-directed mutagenesis nucSTB

T168AF GCGTGGAGCAGCTGGCCCGCTACCTCGAGTTGC Site-directed mutagenesis nucSTB

T168AR GCAACTCGAGGTAGCGGGCCAGCTGCTCCACGC Site-directed mutagenesis nucSTB

K184EF GTGCTCGCGCCGGTCGAGGGGGTGTTTGCCG Site-directed mutagenesis nucSTB

K184ER CGGCAAACACCCCCTCGACCGGCGCGAGCAC Site-directed mutagenesis nucSTB

pvv16-seq-fwd CGGTGAGTCGTAGGTCGGGACGG PCR amplification and sequencing

pvv16-seq-rev TGCCTGGCAGTCGATCGTACGCTAG PCR amplification and sequencing

19

Supplementary Methods.

Generation of a ΔnucS knockout mutant in M. smegmatis.

To generate a ΔnucS knockout mutant in M. smegmatis, a 1.0 kb PstI-HindIII upstream and HindIII-

SalI downstream fragments of nucS were amplified by PCR, using delnucSm 5F, 5R, 3F and 3R

primers, and cloned in-frame into the p2NIL vector. Then, a pGOAL19 PacI-cassette, carrying a β-

galactosidase lacZ gene, a hygromycin-resistance hyg gene and a sacB gene, that confers sucrose

sensitivity, was also inserted into the p2NIL vector. The resulting plasmid p2NIL-ΔnucSSm,

harbouring an in-frame deletion of the target gene (lacking ≈ 95% of the gene sequence), was

electroporated into M. smegmatis mc2 155. Cells were plated on Middlebrook 7H10 agar-X-gal

(100 µg ml-1) plus kanamycin (25 µg ml-1) and hygromycin (50 µg ml-1) and incubated for 4-5 days

at 37ºC. Single-crossover merodiploid clones were grown in 7H9 broth without antibiotics to allow

a second crossover event. Cultures were diluted and counter-selected on Middlebrook 7H10-X-gal

(100 µg ml-1) plates containing 2% sucrose and incubated 4-5 days at 37ºC. Double-crossover

clones were tested for kanamycin and hygromycin susceptibility to confirm the loss of the plasmid.

Finally, M. smegmatis mc2 155 ΔnucS colonies were tested by PCR and sequencing to verify the

unmarked nucS deletion.

Complementation of the M. smegmatis ΔnucS mutant.

For complementation with nucS from M. smegmatis mc2 155, the MSMEG_4923 gene (nucSSm),

including its own 46-bp promoter region, was amplified by PCR with primers compnucSmF and

compnucSmR primers, digested with EcoRI and HindIII and cloned into the integrative vector

pMV361, rendering the complementation vector pMV-nucSSm. Similarly, for complementation with

nucS from M. tuberculosis, the wild-type full-length MT_1321 gene from CDC1551 control strain

(nucSTB), with its own 61-pb upstream promoter region, was amplified by PCR, using compnucTBF

and compnucTBR primers, cloned into the pMV361 vector to generate the complementation vector,

pMV-nucSTB. Putative complemented mutants were obtained upon electroporation of the plasmids

into M. smegmatis mc2 155 ΔnucS and incubation of the plated samples on Middlebrook 7H10 agar

plus kanamycin (25 µg ml-1) for 3-5 days at 37ºC. Finally, M. smegmatis mc2 155 ΔnucS

complemented mutants were analysed by PCR and sequencing to verify the proper insertion of the

genes.

20

Generation of ΔnucS S. coelicolor knockout mutant and complementation.

pIJ6650 vector was used to clone in-frame two 1.5 kb DNA fragments, one HindIII-BamHI nucS

upstream fragment plus one BamHI-EcoRV nucS downstream fragment, previously amplified by

PCR (primers delnucSco 5F, 5R, 3F and 3R). E. coli ET12567 (pUZ8002) was transformed with

pIJ-ΔnucSSco (containing the in-frame deletion of the nucSSco gene) and conjugated with S.

coelicolor A3(2) M145. Following the isolation of apramycin resistant single-crossover, putative

double-crossover mutants were isolated and the unmarked deletion of the nucSSco gene was verified

by PCR. S. coelicolor ΔnucS was complemented with a wild-type copy of nucSSco cloned in

pSET152. pSET152 is a non-replicative plasmid in Streptomyces that carries the attP site and the

integrase gene of the C31 phage and consequently can integrate into the attB site 2 in the

chromosome of Streptomyces. nucSSco gene with its own promotor was amplified by PCR with

primers compnucScoF and compnucScoR, cloned into pSET152 using EcoRI and BamHI sites to

give the pSET-nucSSco plasmid that was introduced into E. coli ET12567 (pUZ8002) by

transformation. Finally, pSET-nucSSco from E. coli ET12567 (pUZ8002, pSET-nucSSco) was

introduced into S. coelicolor ΔnucS by conjugation in order to integrate the construction into the

chromosome.

Construction of pRhomyco plasmids.

To create a template for measuring homologous and homeologous recombination in M. smegmatis,

we designed a recombination assay based on the hygromycin-resistance (Hyg-R) gene hyg, using

the integrative plasmid pMV361. For pRhomyco 100%, a fragment denominated hyg 3’ (from

nucleotide 195 of the coding sequence to the stop codon) of the hyg gene, was PCR amplified from

pRAM vector, using rechyg3F and rechyg3R primers and digested with EcoRV/SpeI to be cloned in

StuI/SpeI targets of pMV361. Additionally, a fragment denominated hyg 5´ containing 711 bp

(from the start codon of the hyg gene to nucleotide 711) was PCR amplified and cloned using

PvuII/HindIII with rechyg5F and rechyg5R primers. Both fragments share two overlapping hyg

fragments of 517 bp (nucleotide 195 to 711). The homeologous recombination vectors, named

pRhomyco 95%, 90% and 85%, were constructed by replacing the overlapping 517-bp fragment of

hyg 5´ for synthetic fragments that have 95%, 90% or 85% sequence similarity to the original hyg

fragment (Supplementary Fig. 2). First, a common fragment to the three of them (from nucleotide 1

to 193 of hyg) was PCR-amplified with rechyg5F and rechyg5intR primers and cloned using

PvuII/BclI. Then, each synthetic fragment was PCR amplified and cloned using BclI/NheI with

rec95-BclIF and rec95-NheIR primers, in the case of pRhomyco 95%. For pRhomyco 90% and

21

85%, the variable fragment was cloned using BclI/PcI-BspHI with rec90-85-BclIF and rec90-85-

PciIR primers.

Computational analyses.

A summary of all the computational approaches conducted is depicted in Supplementary Fig. 4. For

NucS, we used the structure-based alignment of bacterial and archaeal NucS (Supplementary Fig.

3). Then we followed the procedure depicted in Supplementary Fig. 4 to conduct domain analyses

using sequences.

Using the different defined regions by structural bioinformatics, we first conducted sequence

searches against the large database. We made non-redundant alignments of the N-terminal region

(containing 63 sequences) and the C-terminal region (containing 39 sequences) and built profile

hidden Markov models (HMMs). These profiles were searched using pHMMER 3 against the large

References Proteomes file where additional proteins containing CT and NT regions in alternative

proteins were found. While the NT region was found in alternative archaeal proteins (we selected

two after checking the alignment F7PKA0_9EURY and L0I7R5_HALRX (DUF91, PF01939), the

CT region was found not only in additional prokaryotic groups, but also in eukaryotic sequences

(I1F1Q3_AMPQE, I1F1Q5_AMPQE, B3SFT8_TRIAD, B9T981_RICCO, and

A0A015J6U4_9GLOM). With the exception of A0A015J6U4, and B9T981, the matching regions

are located within a DUF1016 domain (uncharacterized). We checked the alignments to confirm

that the catalytic residues, as described in the structure of P. abyssi 1, were conserved.

We next searched the PFAM database with NUCS_MYCTU and found a domain, PF01939, which

was trained in three NucS full sequences. From the PF01939 domain (539 sequences in 464

species), only 368 entries in 363 (archaeal and bacterial) species gave a match with the full protein

(Supplementary data file 1).

To identify MutS and MutL in bacterial and archaeal species, we generated HMMs from different

proteins, which would capture most of the bacterial and archaeal diversity (Supplementary Fig. 4).

The MutS profile was trained with the following sequences (MUTS_ECOLI, C1F256_ACIC5,

MUTS_AQUAE, MUTS_BACTN, MUTS_CHLPN, MUTS_CHLTE, MUTS_CHLAA,

WP_027388865.1, MUTS_PROM3, B5YE41_DICT6, MUTS_BACSU, MUTS_STAAM,

MUTS_CLOTE, MUTS_FUSNN, C1AEM6_GEMAT, A6DUF8_9BACT, MUTS_RHOBA,

MUTS_BRUME, MUTS_NEIMA, MUTS_SALTY, MUTS_VIBC3, MUTS_PSEAE,

MUTS_DESVH, MUTS_TREPA, A0A075WU36_9BACT, MUTS_THEMA, WP_009958798.1),

while the MutL profile was trained with the following sequences (MUTL_ACIC5,

MUTL_AQUAE, MUTL_BACTN, MUTL_CHLPN, MUTL_CHLTE, A9WJ86_CHLAA,

22

MUTL_DEIRA, MUTL_DICT6, D9S9H6_FIBSS, MUTL_BACSU, MUTL_STAAM,

MUTL_CLOTE, Q8RG56_FUSNN, MUTL_GEMAT, A6DHB3_9BACT, Q7UMZ3_RHOBA,

MUTL_BRUME, MUTL_NEIMA, A0A0C5TQ33_SALTM, MUTL_VIBCH, MUTL_PSEAE,

Q72ET5_DESVH, MUTL_TREPA, A0A075WSF3_9BACT, MUTL_THEMA, and

MUTL_ECOLI). The models were searched against each reference proteome where bit scores <50

were discarded to filter and analyze the results. Only full proteins were retrieved by forcing a length

> 75%, so partial hits would be excluded.

For actinobacterial and archaeal species not showing NucS, searches in all the particular genomes

(only for complete genomes) were done using translated searches against their genomes using

tblastn with NucS_MYCTU, MutS_ECOLI, and MUTL_ECOLI for actinobacterial, and

NucS_PYRAB, MutS_HALMA, and MUTL_HALSA for archaeal proteins.

23

References for Supplementary Information

1 Ren, B. et al. Structure and function of a novel endonuclease acting on branched DNA substrates. The EMBO journal 28, 2479-‐2489, doi:emboj2009192 [pii] 10.1038/emboj.2009.192 (2009).

2 Bierman, M. et al. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116, 43-‐49 (1992).

3 Eddy, S. R. A new generation of homology search tools based on probabilistic inference. Genome Inform 23, 205-‐211 (2009).

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