TABLE 1.
Strains and plasmids used in this studya
| Strain or plasmid | Characteristics | Source or reference |
|---|---|---|
| Strains | ||
| E. coli DH5α | F− endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 hsdR17 (rK− mK+) λ− | TaKaRa |
| E. coli MG1655 | K-12; F− λ− rph-1 | CGSC 6300 |
| Streptococcus pyogenes MGAS5005 | M1 serotype, wild type | ATCC BAA-947 |
| Tatumella citrea | ||
| DSM 13699 | DSMZ | |
| MGly1 | MG1655 ΔcadA::cat | This study |
| MGly2 | MGly1 Δcat::cadAp | This study |
| 1655ΔcadA | MG1655 ΔcadA | This study |
| 1655ΔcadAΔmaeAΔmaeB | MG1655 ΔcadA ΔmaeA ΔmaeB | This study |
| 1655ΔmaeAΔmaeB | MG1655 ΔmaeA ΔmaeB | This study |
| 1655ΔyjcS::ybaS | MG1655 ΔyjcS::ybaS | This study |
| 1655ΔyjcS::evgAS | MG1655 ΔyjcS::evgAS | This study |
| 1655ΔmaeB::gltPΔmaeA | MG1655 ΔmaeB::gltP ΔmaeA | This study |
| 13699ΔtkrA | DSM 13699 ΔtkrA | This study |
| 13699ΔtkrAΔglk | DSM 13699 ΔtkrA Δglk | This study |
| General plasmids | ||
| pKD46 | repA101(Ts) bla araC ParaB-Red | 13 |
| pSU2718 | p15A cat | 52 |
| pTrc99A | pMB1 bla lacIq | 53 |
| pIJ778 | bla aadA FRT | 54 |
| pKD46K | repA101(Ts) kan araC ParaB-Red | 20 |
| pTrc99A-spec | pMB1 aadA lacIq | This study |
| Crispr-Cas9 system plasmids | ||
| pCas series | ||
| pCB001 | p15A cm Pcas-cas9 | This study |
| pCas | repA101(Ts) kan Pcas-cas9 ParaB-Red lacIq Ptrc-sgRNA-pMB1 | This study |
| pCasΔcas9 | repA101(Ts) kan ParaB-Red lacIq Ptrc-sgRNA-pMB1 | This study |
| pTarget series harboring sgRNAs, with or without donor DNAs | ||
| pTargetF-pMB1 | pMB1 aadA sgRNA-pMB1 | This study |
| pTargetF-cadA | pMB1 aadA sgRNA-cadA | This study |
| pTargetF-kefB | pMB1 aadA sgRNA-kefB | This study |
| pTargetF-yjcS | pMB1 aadA sgRNA-yjcS | This study |
| pTargetF-cat | pMB1 aadA sgRNA-cat | This study |
| pTargetF-kefB-yjcS | pMB1 aadA sgRNA-kefB, sgRNA-yjcS | This study |
| pTargetF-cadAp | pMB1 aadA sgRNA-cadAp | This study |
| pTargetTΔR-ΔcadAp | pMB1 aadA ΔcadAp (430 bp) | This study |
| pTargetT-ΔcadAp | pMB1 aadA sgRNA-cadAp ΔcadAp (430 bp) | This study |
| pTargetT-ΔcadA | pMB1 aadA sgRNA-cadA ΔcadA (804 bp) | This study |
| pTargetT-ΔmaeA | pMB1 aadA, sgRNA-maeA ΔmaeA (708 bp) | This study |
| pTargetT-ΔmaeB | pMB1 aadA sgRNA-maeB ΔmaeB (829 bp) | This study |
| pTargetT-ΔmaeAΔmaeB | pMB1 aadA sgRNA-maeA, ΔmaeA (708 bp) sgRNA-maeB ΔmaeB (829 bp) | This study |
| pTargetT-ΔcadAΔmaeAΔmaeB | pMB1 aadA sgRNA-cadA ΔcadA (804 bp) sgRNA-maeA ΔmaeA (708 bp) sgRNA-maeB ΔmaeB (829 bp) | This study |
| pTargetT-ΔyjcS::ybaS | pMB1 aadA sgRNA-yjcS ΔyjcS (733 bp):: ybaS (1.3 kb) | This study |
| pTargetT-ΔyjcS::evgAS | pMB1 aadA sgRNA-yjcS ΔyjcS (733bp):: evgAS (4.5 kb) | This study |
| pTargetT-ΔmaeB::gltP | pMB1 aadA sgRNA-maeB ΔmaeB (829 bp)::gltP (1.7 kb) | This study |
| pTargetT-ΔmaeB::gltP ΔmaeA | pMB1 aadA sgRNA-maeB ΔmaeB (829 bp)::gltP (1.7 kb) sgRNA-maeA ΔmaeA (708 bp) | This study |
| pTargetT-ΔtkrA | pMB1 aadA sgRNA-tkrA ΔtkrA (978 bp) | This study |
| pTargetT-Δglk | pMB1 aadA sgRNA-glk Δglk (963 bp) | This study |
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.