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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 20 NucS 15 75 1 2 3 200 A B 10 kDa 150 100 dsDNA
Transcript

  • 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 nucS 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 -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 -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%

    Large DB including 3,942 Ref Proteomes

    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

    Large DB including 3,942 Ref Proteomes

    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_ta

    x_180.str.F0310

    Natronobacterium

    _gregoryi_ATCC

    .43098

    Micro

    bacte

    rium_

    hydro

    carbo

    noxyd

    ans

    Corynebacterium_jeikeium.st.K411

    Aeropyrum_pernix_ATCC.700893

    Halobacterium

    _sp._DL1

    Mycob

    acteriu

    m_abs

    cessus

    _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

    Noca

    rdia_

    cyria

    cigeo

    rgica

    .st.G

    UH-2

    Saccharomonospora_glauca_K62

    Mycobacterium

    _parascrofulac

    eum_ATCC.BA

    A-614

    Stre

    ptom

    yces

    _sp.

    _WM

    6378

    Gardne

    rella_vag

    inalis_A

    TCC.140

    19

    Rhodococcus_jostii.st.R

    HA1

    Pyro

    bacu

    lum

    _aer

    ophi

    lum

    _ATC

    C.51

    768

    Stre

    ptom

    yces

    _rub

    ello

    mur

    inus

    Actinomyces_m

    assiliensis_F04

    89

    Rhodococcus_aetherivorans

    Mycobacte

    rium_sinen

    se.st.JDM6

    01

    Actinomyces_sp.oral_tax_1

    78.str.F0338

    Rhodococcus_sp._B

    7740

    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

    ptomy

    ces_

    rimos

    us_s

    ubsp

    ._rim

    osus

    _ATC

    C.10

    970

    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

    Mycoba

    cterium

    _hassia

    cum_DS

    M.4419

    9

    Streptomyces_cattleya_ATCC.35852

    Rhodococcus_equi_ATCC.3370

    7

    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

    Mycobacteriu

    m_paratuber

    culosis_ATCC

    .BAA-968

    Natrinem

    a_sp.st.J7-2

    Halorubrum

    _lacusprofundi_ATCC

    .49239

    Noca

    rdia_

    nova

    _SH2

    2a

    2290

    07.

    CCT

    A_mu

    ranil

    as_

    muir

    etca

    bola

    H

    Microb

    acteriu

    m_lae

    vanifor

    mans_

    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_s

    p._oral_tax_17

    5.str.F0384

    Stre

    ptomy

    ces_

    grise

    us_s

    ubsp

    ._gris

    eus.s

    t.JCM

    _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

    Bifidob

    acteriu

    m_sca

    rdovii

    Scardov

    ia_wiggs

    iae_F04

    24

    Marinitherm

    us_hydrothermalis.st.DSM

    .14884

    Sanguibacter_keddieii_ATCC.51767

    Mobiluncus_mulieris_ATCC.352

    39

    Actin

    obac

    teria

    _bac

    teriu

    m_I

    MCC

    2625

    6Actinom

    yces_graeven

    itzii_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

    Bifidobacte

    rium_gallic

    um_DSM.2

    0093

    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

    Dietz

    ia_cin

    name

    a_P4

    Deinococcus_proteolyticus_ATCC.35074

    Micro

    bacte

    rium_

    oxyd

    ans

    Corynebacterium_vitaeruminis_DSM.20294

    Noca

    rdia_

    farcin

    ica.st

    .IFM_

    1015

    2

    Nocardia_sp._NRRL_S-836

    Mycoba

    cterium

    _thermo

    resistib

    ile_ATC

    C.1952

    7

    Corynebacterium_diphtheriae_ATCC.700971

    Actinoplanes_sp._N902-109

    Bifidob

    acterium

    _longum

    .st.NCC

    _2705

    Stre

    ptom

    yces

    _sp.

    _NRR

    L_F-

    6602

    Mycobacteriu

    m_haemop

    hilum

    Gordo

    nia_po

    lyisopr

    enivor

    ans.st.

    DSM.4

    4266

    Pyrolobus_fumarii.DSM

    .11204

    Stre

    ptom

    yces

    _sp.

    _PB

    H53

    Bifidobacter

    ium_bifidum

    _BGN4

    Dermacoccus_nishinomiyaensis

    misce_Crenarchaeota_group-1_archaeon_SG8-32-1

    Stre

    ptom

    yces

    _dav

    awen

    sis_

    JCM

    _491

    3

    Bifidobac

    terium_a

    ngulatum

    _DSM.20

    098

    Stre

    ptom

    yces

    _xia

    men

    ensis

    Microb

    acteri

    um_ke

    tosire

    ducen

    s

    Mycobacte

    rium_vanb

    aalenii.st.D

    SM.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

    Mycobacteriu

    m_leprae.st.T

    N

    Gordo

    nia_sp

    uti_NB

    RC.10

    0414

    Mobiluncus_curtisii_ATCC.43063

    Mobil

    icocc

    us_p

    elagiu

    s_NB

    RC.10

    4925

    Nakamurella_multipartita_ATCC.700099

    Prop

    ionim

    icrob

    ium_ly

    mpho

    philu

    m_AC

    S-09

    3-V-

    SCH5

    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

    Microb

    acteriu

    m_tric

    hothec

    enolyti

    cum

    Corynebacterium_pseudotuberculosis.st.C231

    Stre

    ptomy

    ces_

    niveu

    s_NC

    IMB_

    1189

    1

    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

    Mycobacte

    rium_obue

    nse

    Bifidob

    acteriu

    m_lon

    gum_su

    bsp.lon

    gum_F8

    Xylanimonas_cellulosilytica.st.DSM_15894

    Mycobacterium_tu

    berculosis_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

    Mycobac

    terium_e

    lephantis

    Deinococcus_gobiensis.st.DSM_21396

    Corynebacterium_xerosis

    Microb

    acteri

    um_ch

    ocolatu

    m

    Mycoba

    cterium_

    smegma

    tis_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

    Segn

    iliparu

    s_rot

    undu

    s_AT

    CC.BA

    A-972

    Jonesia_denitrificans_ATCC.14870

    Gordo

    nia_si

    hwen

    sis_N

    BRC.1

    0823

    6

    69781.M

    SD.ts.ilagtoej_succocilaklala

    H

    Corynebacterium_falsenii_DSM.44353

    Rhodococcus_spRD6.2

    Bifidobacte

    rium_dentiu

    m_ATCC.27

    534

    Bifidoba

    cterium

    _thermo

    philum_R

    BL67

    Halopiger_xanaduensis.st.D

    SM.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

    Micro

    bacte

    rium_

    sp._S

    A39

    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

    Brac

    hyba

    cteriu

    m_sp

    ._SW

    0106

    -09

    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

    Mycoba

    cterium

    _xenop

    i_RIVM

    700367

    Saccharomonospora_marina_XMU15

    Bifidob

    acteriu

    m_ast

    eroide

    s.st.P

    RL201

    1Th

    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

    Bifidobacte

    rium_adole

    scentis_AT

    CC.15703

    Micro

    bacte

    rium_

    folior

    um

    Catenulispora_acidiphila.st.DSM_44928

    Micro

    bacte

    rium_

    azad

    irach

    tae

    Lechevalieria_aerocolonigenes

    Amycolatopsis_mediterranei.st.S699

    Corynebacterium_sp._ATCC.6931

    Gordo

    nia_rh

    izosph

    era_N

    BRC.1

    6068

    Thermosphaera_aggregans.st.D

    SM.11486

    Salinispora_tropica_ATCC.BAA-916

    I4F2P8 acti 477641

    Mycobacterium_m

    arinum_ATCC.BA

    A-535

    halophilic_archaeon_DL31

    Streptomyces_katrae

    Streptomyces_sp._W

    M6372

    Bifidoba

    cterium

    _breve

    _DSM.2

    0213

    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_AT

    CC.83

    68

    Thermofilum

    _pendens.st.Hrk_5

    Corynebacterium_variabile.st.DSM_44702

    Frankia_sp.st.EuI1c

    Bifidobacte

    rium_kash

    iwanohens

    e_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_ac

    idipr

    opion

    ici_A

    TCC.

    4875

    Gordo

    nia_a

    marae

    _NBR

    C.155

    30

    Stre

    ptom

    yces

    _inc

    arna

    tus

    Mycobacte

    rium_neoa

    urum_VKM

    _Ac-1815D

    Actinoplanes_sp._ATCC.31044

    Mycobact

    erium_sp.

    st.KMS)

    Patulibacter_medicamentivorans

    Noca

    rdia_

    brasili

    ensis

    _ATC

    C.700

    358

    Strep

    tomyc

    es_p

    raten

    sis_A

    TCC.

    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

    Kineo

    spha

    era_li

    mosa

    _NBR

    C.10

    0340

    Stre

    ptomy

    ces_

    prist

    inaes

    pirali

    s_AT

    CC.25

    486

    Nocardioides_sp._CF8

    Thermobispora_bispora_ATCC.19993

    Staphylothermus_m

    arinus_ATCC

    .43588

    Kocuria_rhizophila_ATCC.9341

    Actinomyces

    _urogenitalis_

    DSM.15434

    Corynebacterium_glyciniphilum_AJ_3170

    Gordo

    nia_sp

    ._KTR

    9

    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

    Actinomyces_sp

    .oral_tax_448.str.

    F0400

    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

    Strep

    tomyc

    es_s

    p.st.S

    irexA

    A-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

    _AGR

    0001

    Brac

    hyba

    cteriu

    m_fae

    cium_

    ATCC

    .4388

    5

    Micro

    bacte

    rium_

    testac

    eum.

    st.StL

    B037

    L0J2N8

    acti 21

    2767

    Stre

    ptom

    yces

    _sp.

    st.S

    PB07

    4

    Actinoplanes_missouriensis_ATCC.14538

    Actinomyces_turicen

    sis_ACS-279-V-Col4

    Mycoba

    cterium

    _sp._VK

    M_Ac-18

    17D

    Bifidob

    acteriu

    m_ani

    malis_

    subsp.

    _lactis

    .st.AD

    011

    Streptomyces_sp._W

    M4235

    Gardnere

    lla_vagin

    alis_JCP

    8481B

    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

    Micro

    bacte

    rium_

    sp._A

    g1

    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

    Microb

    acteriu

    m_gin

    sengis

    oli

    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

    Gordo

    nia_br

    onchia

    lis_AT

    CC.25

    592

    Bifidobac

    terium_p

    seudolon

    gum_PV

    8-2

    Stre

    ptom

    yces

    _alb

    us_A

    TCC.

    2183

    8

    Sulfo

    lobu

    s_ac

    idoc

    alda

    rius_

    ATCC

    .339

    09

    Saccharomonospora_viridis_ATCC.15386

    Strep

    tomyc

    es_c

    lavuli

    gerus

    _ATC

    C.27

    064

    Frankia_sp.st.CcI3

    Isoptericola_variabilis.st.225

    540M_sucaitnaruaoesirg_secy

    motpertS

    Sacc

    haro

    thrix

    _sp.

    _ST-

    888

    Actinoplanes_friuliensis_DSM.7358

    Mycobac

    terium_r

    hodesiae

    .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

    Scardov

    ia_inop

    inata_F

    0304

    Met

    hano

    bact

    eriu

    m_p

    alud

    is.s

    t.DSM

    .258

    20

    Mycob

    acteriu

    m_sp.

    _EPa45

    Actinomyces_neui

    i_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-

    Marin

    ither

    mus h

    ydro

    therm

    alis.s

    t.DSM

    1488

    4169

    -Coryne

    bacteriu

    m camp

    orealens

    is

    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 1

    05200

    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-Halo

    arcula m

    arismortu

    i.st.ATCC

    .43049

    175-Cory

    nebacteri

    um sp.AT

    CC.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-Co

    ryneba

    cterium

    epide

    rmidic

    anis

    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-Ha

    lalkalic

    occus j

    eotgali.s

    t.DSM 1

    8796

    162-Co

    ryneba

    cterium

    efficie

    ns.st.D

    SM 44

    549

    181-Coryne

    bacterium l

    ipophiloflav

    um.DSM.442

    91

    333-

    Micr

    omon

    ospo

    ra s

    p.NR

    RL B

    -168

    02

    387-Halo

    quadratum

    walsbyi.s

    t.DSM 16

    790

    153-C

    oryne

    bacte

    rium

    variab

    ile.st.D

    SM 44

    702

    360-M

    ethan

    otherm

    obac

    ter th

    ermau

    totrop

    hicus

    .st.AT

    CC.29

    096

    161-Co

    ryneba

    cterium

    glutam

    icum.s

    t.ATCC

    .13032

    346-

    Segn

    ilipar

    us ro

    tundu

    s.st.A

    TCC.

    BAA-

    972

    390-Natron

    obacterium

    gregoryi.s

    t.ATCC.43

    098

    278-Saccharomonospora glauca K62

    225-Arthrobacter arilaitensis.st.DSM 16368

    39-Vibrio campbellii.st.HY01

    173-Cory

    nebacteri

    um aurim

    ucosum.s

    t.ATCC.7

    00975

    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

    419-Clostridium sp.CAG:433

    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-Co

    ryneba

    cterium

    sp.KP

    L1989

    420-Clostridium sp.CAG:433

    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-Halo

    geometricu

    m borinqu

    ense.st.AT

    CC.70027

    4

    329-

    Salin

    ispor

    a tro

    pica

    .st.A

    TCC.

    BAA-

    916

    68-Thermobispora bispora.st.ATCC.19993

    46-Deinococcus maricopensis.st.DSM 21211

    369-Th

    ermofil

    um pe

    ndens.

    st.Hrk 5

    102-

    Stre

    ptom

    yces

    rube

    llom

    urin

    us

    215-Microbacterium sp.SA39406-Pyrococcus

    abyssi.st.GE5

    34-Caldivirga maquilingensis.st.ATCC.700844

    154-C

    oryne

    bacte

    rium

    jeikeiu

    m.st.K

    411

    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 k

    odakarensis.st.ATCC

    .BAA-918

    182-Coryneb

    acterium ure

    icelerivorans

    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-Cory

    nebacteri

    um casei

    LMG S-1

    9264

    185-Streptospo

    rangium roseum

    .st.ATCC.1242

    8

    287-Actinomyces sp.oral taxon 180 str.F0310

    80-Streptomyces sp.W

    M6372

    376-Na

    tronoba

    cterium

    gregor

    yi.st.AT

    CC.43

    098

    396-Natrialba

    magadii.st.AT

    CC.43099

    43-Deinococcus gobiensis.st.DSM 21396

    281-Actinomyces graevenitzii C83

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

    373-Ca

    ndidat

    us Ma

    gnetob

    acteriu

    m bava

    ricum

    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-Co

    ryneba

    cterium

    kutsch

    eri

    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-Ha

    lobacter

    ium sal

    inarum.

    st.ATCC

    .700922

    53-Frankia sp.st.EuI1c

    151-C

    oryne

    bacte

    rium

    false

    nii.DS

    M.44

    353

    32-Ignisphaera aggregans.st.DSM 17230

    37-Paraglaciecola arctica BSs20135

    49-Frankia alni.st.ACN14a

    143-S

    trepto

    myce

    s cae

    lestis

    404-Thermococcus barop

    hilus.st.DSM 11836

    277-Nocardia nova SH22a

    320-

    Geo

    derm

    atop

    hilu

    s ob

    scur

    us.s

    t.ATC

    C.2

    5078

    395-Haloterr

    igena turkme

    nica.st.ATCC

    .51198

    168-Co

    rynebac

    terium m

    aris.DS

    M.4519

    0

    125-

    Stre

    ptom

    yces

    col

    linus

    .st.D

    SM 4

    0733

    391-Halopig

    er xanadu

    ensis.st.DS

    M 18323

    232-Arthrobacter sp.st.FB24

    411-Metagenomes

    296-Scardovia inopinata F0304

    400-Crenarchaeot

    a group-1 archae

    on SG8-32-1

    11-Synechococcus sp.ATCC.27264

    214-Microbacterium hydrocarbonoxydans

    230-Arthrobacter globiformis NBRC 12137

    195-Kineococcus radiotolerans.st.ATC

    C.BAA-149

    19-Chryseobacterium sp.ERMR1:04

    86-Streptomyces sp.PVA 94-07

    370-Lo

    kiarch

    aeum s

    p.GC1

    4 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-Crenarchae

    ota group-6 arch

    aeon AD8-1

    299-Bifidobacterium gallicum

    .DSM

    .20093

    75-Streptomyces sp.M

    g1

    377-Ha

    lobacter

    ium sp.

    DL1

    72-Nocardiopsis sp.NRRL B-16309

    136-

    Stre

    ptomy

    ces g

    lauce

    scen

    s

    205-Cellulomonas gilvus.st.ATCC.13127

    84-Streptomyces venezuelae.st.ATC

    C.10712

    291-Catenulispora acidiphila.st.D

    SM 44928

    380-Na

    tronomo

    nas mo

    olapens

    is.st.DS

    M 1867

    4

    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

    diphth

    eriae

    .st.AT

    CC.70

    0971

    321-

    Blas

    toco

    ccus

    sax

    obsi

    dens

    .st.D

    D2

    122-

    Stre

    ptom

    yces

    sp.

    PBH5

    3

    146-B

    reviba

    cteriu

    m mc

    brelln

    eri AT

    CC.49

    030

    4-Methanococcoides methylutens MM1

    386-Halo

    ferax volc

    anii.st.AT

    CC.29605

    190-Intrasporangium

    calvum.st.ATCC.235

    52

    45-Deinococcus proteolyticus.st.ATCC.35074

    179-Coryne

    bacterium

    marinum.D

    SM.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

    288-Actinomyces sp.oral taxon 178 str.F0338

    349-

    Aero

    pyru

    m pe

    rnix.

    st.AT

    CC.70

    0893

    273-Mycobacterium

    hassiacum.DSM

    .44199

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

    405-Thermococcus litoralis.s

    t.ATCC.51850

    283-Actinomyces neuii BVS029A5

    108-

    Stre

    ptom

    yces

    vie

    tnam

    ensi

    s

    223-Microbacterium chocolatum

    135-

    Stre

    ptomy

    ces d

    avaw

    ensis

    JCM

    4913

    359-M

    ethan

    obac

    terium

    sp.M

    B1

    133-

    Stre

    ptom

    yces

    sp.e

    14

    91-Streptomyces clavuligerus.st.ATC

    C.27064

    372-Ca

    ndidat

    us Ma

    gnetob

    acteriu

    m bava

    ricum

    279-Saccharomonospora m

    arina XMU15

    20A sucidyl secymotpertS-69

    63-Saccharothrix espanaensis.st.ATCC.51144

    152-C

    oryne

    bacte

    rium

    glycin

    iphilum

    AJ 31

    70

    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-Cor

    ynebacte

    rium stria

    tum ATC

    C.6940

    167-Co

    rynebac

    terium g

    enitaliu

    m ATCC

    .33030

    352-T

    herm

    oglad

    ius ce

    llulol

    yticu

    s.st.1

    633 371

    -Lokia

    rchaeu

    m sp.G

    C14 7

    5

    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-Co

    ryneba

    cterium

    mustel

    ae

    69-Thermom

    onospora curvata.st.ATCC.19995

    139-

    Stre

    ptomy

    ces s

    p.NRR

    L WC-

    3618

    89-Streptomyces sp.st.SirexAA-E

    121-

    Stre

    ptom

    yces

    sp.

    MUS

    C119

    T

    365-M

    ethan

    ospha

    era sta

    dtman

    ae.st.

    ATCC

    .4302

    1

    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-Co

    ryneba

    cterium

    glucur

    onolyti

    cum AT

    CC.51

    866

    227-Arthrobacter chlorophenolicus.st.ATCC.700700

    316-

    Amyc

    olat

    opsi

    s or

    ient

    alis

    HC

    CB1

    0007

    140-S

    trepto

    myce

    s virid

    ochro

    moge

    nes

    183-Coryneb

    acterium test

    udinoris

    103-

    Sacc

    haro

    thrix

    sp.

    ST-8

    88

    145-S

    trepto

    myce

    s sp.W

    M637

    8

    149-C

    oryne

    bacte

    rium

    resiste

    ns.st

    .DSM

    4510

    0

    355-H

    ypert

    herm

    us bu

    tylicu

    s.st.D

    SM 54

    5655-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 Ca

    ldiarchaeum subte

    rraneum

    394-Natrono

    coccus occu

    ltus SP4

    353-T

    herm

    osph

    aera

    aggre

    gans

    .st.D

    SM 11

    486

    224-Kocuria sp.UCD-OTCP

    158-Co

    ryneba

    cterium

    argent

    oraten

    se.DS

    M.4420

    2

    315-

    Stre

    ptom

    yces

    sp.

    AA4

    218-Microbacterium foliorum

    967.ps secymotpertS-59

    354-a

    rchae

    on G

    W20

    11 AR

    10

    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

    myce

    s 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 N

    BRC 104925

    82-Streptomyces niveus N

    CIM

    B 11891

    138-

    Stre

    ptomy

    ces s

    cabie

    i.st.8

    7.22

    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

    myce

    s gha

    naen

    sis AT

    CC.14

    672

    367-M

    ethano

    pyrus

    kandle

    ri.st.A

    V19

    1000

    RGA

    sut

    arua

    sec

    ymo

    tpert

    S-49

    12-Desulfobacterium autotrophicum.st.ATCC.43914

    229-Brachybacterium sp.SW0106-09

    87-Streptomyces sp.N

    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-Coryne

    bacterium

    humireduc

    ens NBRC

    106098

    144-S

    trepto

    myce

    s cya

    neog

    riseu

    s sub

    sp.no

    ncya

    noge

    nus

    176-Cory

    nebacteriu

    m doosan

    ense CAU

    212

    177-Coryne

    bacterium

    halotolera

    ns YIM 70

    093

    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-M

    ethan

    obac

    terium

    palud

    is.st.D

    SM 25

    820

    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 smi

    thii.st

    .PS

    166-Co

    rynebac

    terium p

    seudotu

    berculo

    sis.st.C

    231

    259-Mycobacterium kansasii ATCC.12478

    282-Actinomyces sp.oral taxon 448 str.F0400

    13-Streptomyces viridochromogenes

    381-Ha

    lobacter

    ium sp.

    DL1

    403-Thermococcus ga

    mmatolerans.st.DSM 1

    5229

    184-Coryneba

    cterium utereq

    ui

    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.435

    87

    83-Streptomyces sp.N

    RR

    L F-6491

    350-

    Pyro

    lobus

    fuma

    rii.st.

    DSM

    1120

    4

    385-halo

    philic arc

    haeon DL

    31

    3-Methanolobus psychrophilus R15

    170-Co

    rynebac

    terium s

    p.KPL18

    55

    77-Streptomyces sp.NRRL S-4441

    27-S

    trept

    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-Th

    ermofil

    um sp

    .1910b

    268-Mycobacterium

    vanbaalenii.st.DSM 7251

    398-Halomicro

    bium mukohata

    ei.st.ATCC.700

    874

    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

    matru

    choti

    i ATC

    C.14

    266

    40-Firmicutes bacterium CAG:114

    8F mugnol.psbus

    mugnol muiretcabodifiB-903

    389-Haloru

    brum lacu

    sprofundi.

    st.ATCC.4

    923910

    5-St

    rept

    omyc

    es s

    p.C

    NQ

    -509

    234-Micrococcus luteus.st.ATCC.4698

    361-M

    ethan

    obrev

    ibacte

    r rumi

    nanti

    um.st

    .ATCC

    .3506

    3

    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

    urealy

    ticum.

    st.AT

    CC.43

    042

    382-Ha

    lobacter

    ium sali

    narum.s

    t.ATCC.

    700922

    157-Co

    ryneba

    cterium

    atypic

    um

    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-Halostagn

    icola larsenii X

    H-48

    248-Gordonia amarae NBRC 15530

    303-Bifidobacterium scardovii

    374-Ca

    ndidat

    us Ma

    gnetob

    acteriu

    m bava

    ricum

    134-

    Stre

    ptomy

    ces 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-Natrine

    ma sp.st.J7

    -2

    357-S

    ulfolo

    bus s

    olfata

    ricus

    .st.AT

    CC.35

    092

    18-Amphimedon queenslandica

    383-Nat

    ronomon

    as phara

    onis.st.A

    TCC.356

    78

    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 P

    VAS-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-Natrine

    ma pellirubr

    um.st.DSM

    15624

    412-Thiothrix nivea.DSM.5205

    187-Dermacoccu

    s nishinomiyaens

    is

    31-Pyrobaculum aerophilum.st.ATCC.51768

    311-

    True

    pere

    lla p

    yoge

    nes

    155-Co

    ryneba

    cterium

    kroppe

    nstedt

    ii.st.D

    SM 44

    385

    356-S

    ulfolo

    bus t

    okod

    aii.st

    .DSM

    1699

    3

    180-Coryne

    bacterium i

    mitans

    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-Cory

    nebacteri

    um xeros

    is

    137-

    Actin

    obac

    teria

    bacte

    rium

    OK07

    4

    165-Co

    ryneba

    cterium

    vitaeru

    minis.D

    SM.20

    294

    351-

    Stap

    hylot

    herm

    us m

    arinu

    s.st.A

    TCC.

    4358

    8

    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-M

    ethan

    obact

    erium

    lacus.

    st.AL-2

    1

    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-Rh

    izophag

    us irre

    gularis

    DAOM

    19719

    8w

    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

    208-Actinomyces_sp.oral_taxon_178_str.F0338

    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

    209-Actinomyces_sp.oral_taxon_180_str.F0310

    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

    22-Streptomyces_sp.WM6378

    365-Streptomyces_sp.NRRL_F-6602

    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

    202-Actinomyces_sp.oral_taxon_175_str.F0384

    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

    205-Actinomyces_sp.oral_taxon_448_str.F0400

    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

    20-Streptomyces_sp.WM4235

    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

    3-Streptomyces_sp.NRRL_F-6602

    249-Arthrobacter_sp.st.FB24

    73-Streptomyces_sp.WM6386

    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

    29-Streptomyces_sp.NRRL_F-4428

    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 nucS derivative. B) Specificity

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

    A)

    Position

    Codon change

    Amino acid change

    WT

    nucS

    A 1295 G

    GACGGC

    Asp 432 Gly

    0

    1

    C 1324 T

    CACTAC

    His 442 Tyr

    6

    6

    C 1324 G

    CACGAC

    His 442 Asp

    2

    0

    A 1325 G

    CACCGC

    His 442 Arg

    13

    20

    A 1325 C

    CACCCC

    His 442 Pro

    4

    0

    G 1334 A

    CGT CAT

    Arg 445 His

    0

    6

    G 1334 C

    CGTCCT

    Arg 445 Pro

    1

    0

    G 1334 T

    CGTCTT

    Arg 445 Leu

    2

    0

    C 1340 T

    TCGTTG

    Ser 447 Leu

    9

    6

    C 1340 G

    TCGTGG

    Ser 447 Trp

    2

    0

    T 1346 C

    CTGCCG

    Leu 449 Pro

    0

    2

    G 1348 A

    GGCAGC

    Gly 450 Ser

    1

    0

    CCAGCTGTC (1275-1283)

    CCAGCTGTC

    Ser 425 Arg + GlnLeuSer

    (426-428)

    1

    0

    TOTAL

    41

    41

  • 12

    B)

    Mutation WT (%) n=41

    nucS (%) n=41

    G:CA:T 16 (39.0) 18 (43.9) A:TG:C 13 (31.7) 23 (56.1) G:CT:A 2 (4.9) 0 A:TT:A 0 0 G:CC:G 5 (12.2) 0 A:TC: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 AGCAGG 3.49x10-7 2.93-4.0x10-7 83.7

    S54I AGTATT 7.94x10-9 5.49-11.07x10-9 1.9

    A67S GCGTCG 2.78x10-9 1.87-3.84x10-9 0.7

    V69A GTGGCG 8.77x10-9 6.50-11.2x10-9 2.1

    A135S GCGTCG 3.57x10-8 2.77-4.38x10-8 8.6

    R144S CGCAGC 3.16x10-8 2.40-4.0x10-8 7.6

    D162H GACCAC 8.98x10-9 6.42-11.80x10-9 2.2

    T168A ACCGCC 3.96x10-8 3.09-4.81x10-8 9.5

    K184E AAGGAG 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 nucSSm complementation

    CompnucTBR ACTGAAGCTTTCAGAACAGCCGGTACTCGCCGCT nucSTB for nucSSm complementation

    CompnucSmF ACTGGAATTCCCGCGCCAGCGAATTGTCGGCGTTCAT nucSSm for nucSSm complementation

    CompnucSmR CGGCAAGCTTTCAGAAGAGCCGGTACTCGTCGCT nucSSm for nucSSm 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 nucSSco complementation

    CompnucScoR TACGGGATCCTCAGAACAGCCGCAGCTTGTCGTCCTCGATGCC nucSSco for nucSSco 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 nucS knockout mutant in M. smegmatis.

    To generate a nucS 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 -

    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-nucSSm,

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

    at 37C. 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 g ml-1) plates containing 2% sucrose and incubated 4-5 days at 37C. Double-crossover

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

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

    unmarked nucS deletion.

    Complementation of the M. smegmatis nucS 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 nucS and incubation of the plated samples on Middlebrook 7H10 agar

    plus kanamycin (25 g ml-1) for 3-5 days at 37C. Finally, M. smegmatis mc2 155 nucS

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

    genes.

  • 20

    Generation of nucS 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-nucSSco (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 nucS 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 nucS 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


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