TABLE 1

Strains and plasmids used in this studya

Strain or plasmidCharacteristicsSource or reference
Strains
    E. coli DH5αF endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 hsdR17 (rK mK+) λTaKaRa
    E. coli MG1655K-12; F λ rph-1CGSC 6300
    Streptococcus pyogenes MGAS5005M1 serotype, wild typeATCC BAA-947
    Tatumella citrea
        DSM 13699DSMZ
        MGly1MG1655 ΔcadA::catThis study
        MGly2MGly1 Δcat::cadApThis study
            1655ΔcadAMG1655 ΔcadAThis study
            1655ΔcadAΔmaeAΔmaeBMG1655 ΔcadA ΔmaeA ΔmaeBThis study
            1655ΔmaeAΔmaeBMG1655 ΔmaeA ΔmaeBThis study
            1655ΔyjcS::ybaSMG1655 ΔyjcS::ybaSThis study
            1655ΔyjcS::evgASMG1655 ΔyjcS::evgASThis study
            1655ΔmaeB::gltPΔmaeAMG1655 ΔmaeB::gltP ΔmaeAThis study
            13699ΔtkrADSM 13699 ΔtkrAThis study
            13699ΔtkrAΔglkDSM 13699 ΔtkrA ΔglkThis study
General plasmids
    pKD46repA101(Ts) bla araC ParaB-Red13
    pSU2718p15A cat52
    pTrc99ApMB1 bla lacIq53
    pIJ778bla aadA FRT54
    pKD46KrepA101(Ts) kan araC ParaB-Red20
    pTrc99A-specpMB1 aadA lacIqThis study
Crispr-Cas9 system plasmids
    pCas series
        pCB001p15A cm Pcas-cas9This study
        pCasrepA101(Ts) kan Pcas-cas9 ParaB-Red lacIq Ptrc-sgRNA-pMB1This study
        pCasΔcas9repA101(Ts) kan ParaB-Red lacIq Ptrc-sgRNA-pMB1This study
    pTarget series harboring sgRNAs, with or without donor DNAs
        pTargetF-pMB1pMB1 aadA sgRNA-pMB1This study
        pTargetF-cadApMB1 aadA sgRNA-cadAThis study
        pTargetF-kefBpMB1 aadA sgRNA-kefBThis study
        pTargetF-yjcSpMB1 aadA sgRNA-yjcSThis study
        pTargetF-catpMB1 aadA sgRNA-catThis study
        pTargetF-kefB-yjcSpMB1 aadA sgRNA-kefB, sgRNA-yjcSThis study
        pTargetF-cadAppMB1 aadA sgRNA-cadApThis study
        pTargetTΔR-ΔcadAppMB1 aadA ΔcadAp (430 bp)This study
        pTargetT-ΔcadAppMB1 aadA sgRNA-cadAp ΔcadAp (430 bp)This study
        pTargetT-ΔcadApMB1 aadA sgRNA-cadA ΔcadA (804 bp)This study
        pTargetT-ΔmaeApMB1 aadA, sgRNA-maeA ΔmaeA (708 bp)This study
        pTargetT-ΔmaeBpMB1 aadA sgRNA-maeB ΔmaeB (829 bp)This study
        pTargetT-ΔmaeAΔmaeBpMB1 aadA sgRNA-maeA, ΔmaeA (708 bp) sgRNA-maeB ΔmaeB (829 bp)This study
        pTargetT-ΔcadAΔmaeAΔmaeBpMB1 aadA sgRNA-cadA ΔcadA (804 bp) sgRNA-maeA ΔmaeA (708 bp) sgRNA-maeB ΔmaeB (829 bp)This study
        pTargetT-ΔyjcS::ybaSpMB1 aadA sgRNA-yjcS ΔyjcS (733 bp):: ybaS (1.3 kb)This study
        pTargetT-ΔyjcS::evgASpMB1 aadA sgRNA-yjcS ΔyjcS (733bp):: evgAS (4.5 kb)This study
        pTargetT-ΔmaeB::gltPpMB1 aadA sgRNA-maeB ΔmaeB (829 bp)::gltP (1.7 kb)This study
        pTargetT-ΔmaeB::gltP ΔmaeApMB1 aadA sgRNA-maeB ΔmaeB (829 bp)::gltP (1.7 kb) sgRNA-maeA ΔmaeA (708 bp)This study
        pTargetT-ΔtkrApMB1 aadA sgRNA-tkrA ΔtkrA (978 bp)This study
        pTargetT-ΔglkpMB1 aadA sgRNA-glk Δglk (963 bp)This study
  • a bla, ampicillin resistance gene; kan, kanamycin resistance gene; aadA, spectinomycin resistance gene; cat, chloramphenicol resistance gene; Pcas-cas9, the cas9 gene with its native promoter; ParaB-Red, the Red recombination genes with an arabinose-inducible promoter; Ptrc-sgRNA-pMB1, sgRNA with an N20 sequence for targeting the pMB1 region with a trc promoter; sgRNA-cadA, sgRNA with an N20 sequence for targeting the cadA locus; sgRNA-cadAp, sgRNA with an N20 sequence for targeting the partial cadA fragment inserted inside the heterologous cat loci of strain MGly2. ΔcadA (804 bp), editing template with an 804-bp region homologous to the cadA locus; ΔcadAp (430 bp), editing template with a 430-bp region homologous to the partial cadA fragment inserted inside the heterologous cat loci of strain MGly2; ΔmaeA (708 bp), editing template with an 804-bp region homologous to the maeA locus; ΔyjcS (733 bp)::ybaS (1.3 kb), editing template with an 804-bp region homologous to the yjcS locus with a 1.3-kb ybaS insertion; ΔyjcS (733 bp)::evgAS (4.5 kb), editing template with a 733-bp region homologous to the yjcS locus with a 4.5-kb evgAS insertion; ΔmaeB (829 bp)::gltP (1.7 kb), editing template with an 829-bp region homologous to the maeB locus with a 1.7-kb gltP insertion.