TABLE 1.
Strains used in this studya
| Strain | Genotypeb | Construction | Source or reference |
|---|---|---|---|
| ES114 | Wild type | NA | 52 |
| KB2B1 | Wild type | NA | 62, 63 |
| KV1787 | ΔsypG | NA | 8 |
| KV3378 | ΔrscS | NA | 32 |
| KV5367 | ΔsypF | NA | 20 |
| KV6475 | ΔsypG attTn7::sypG-FLAG | NA | 61 |
| KV6527 | attTn7::sypG-D53E-FLAG | NA | 20 |
| KV6659 | ΔsypF attTn7::sypF-FLAG | NA | 20 |
| KV7226 | ΔsypF attTn7::sypF-HPT-FLAG | NA | 20 |
| KV7371 | IG::PsypA-lacZ | NA | 20 |
| KV7964 | ΔhahK:: FRT-Cm | NA | 19 |
| KV8025 | ΔhnoX:: FRT-Erm | NA | 23 |
| KV8079 | ΔsypQ::FRT-Cm IG::PsypA lacZ attTn7::Erm | NA | 19 |
| KV8232 | IG::Ermr-trunc Trimr | NA | 57 |
| KV8484 | ΔhnoX-hahK::FRT-Erm | NA | 23 |
| KV9191 | ΔsypG::FRT-Spec | TT ES114 with SOE product amplified with primers 1223 and 1221 (ES114), primers 2089 and 2090 (pKV521), and primers 2437 and 427 (ES114) | This study |
| KV9501 | ΔrscS::FRT-Spec | TT ES114 with SOE product amplified with primers 2918 and 2919 (ES114), primers 2089 and 2090 (pKV521), and primers 2920 and 2921 (ES114) | This study |
| KV9571 | ΔrscS-PP::FRT-Erm | TT ES114 with SOE product amplified with primers 2918 and 2938 (ES114), primers 2089 and 2090 (pKV494), and primers 2939 and 241 (ES114) | This study |
| KV9622 | IG::PrscS-lacZ | TT KV7371 with SOE product amplified with primers 2185 and 2090 (pKV502), primers 2965 and 2966 (ES114), and primers 2822 and 2876 (KV7371) | This study |
| KV9624 | IG::FRT-Erm | TT ES114 with SOE product amplified with primers 1500 and 2967 (ES114), primers 2089 and 2090 (pKV494), and primers 2968 and 663 (ES114) | This study |
| KV9653 | ΔrscS ΔsypQ::FRT-Cm IG::PsypA lacZ attTn7::Erm | TT KV8079 with gKV9501 | This study |
| KV9782 | rscS-FLAG-FRT-Erm | TT ES114 with SOE product amplified with primers 40 and 3138 (ES114), primers 2354 and 2090 (pKV494), and primers 3139 and 2921 (ES114) | This study |
| KV9942 | ΔrscS::FRT-Spec ΔsypF attTn7::sypF-HPT-FLAG | Derived from KV7226 using KV9501 | This study |
| KV9953 | ΔrscS::FRT ΔsypF attTn7::sypF-HPT-FLAG | Derived from KV9942 | This study |
| KV9968 | ΔhahK::FRT ΔsypF attTn7::sypF-HPT-FLAG | Derived from KV7226 using KV7964 | This study |
| KV10004 | IG::PnrdR-sypA-HA | TT KV8232 with SOE product amplified with primers 2290 and 2090 (pKV506), primers 3083 and 3297 (ES114), and primers 2331 and 1487 (pKV503) | This study |
| KV10019 | IG::PnrdR-sypF-HA | TT KV8232 with SOE product amplified with primers 2290 and 2090 (pKV506), primers 2264 and 2348 (ES114), and primers 2331 and 1487 (pKV503) | This study |
| KV10020 | ΔsypE::FRT-Erm | TT ES114 with SOE product amplified with primers 460 and 2263 (ES114), primers 2089 and 2090 (pKV494), primers 2264 and 425 (ES114) | This study |
| KV10083 | IG::rscS-3′ end-HA | TT KV8232 with SOE products amplified with primers 2331 and 1487 (gKV10004) and primers 2290 and 3292 (gKV9571) | This study |
| KV10101 | IG::PrscS rscS-HA | TT KV10083 with SOE products amplified with primers 2290 and 85 (gKV9624) and primers 2185 and 2090 (pKV502) | This study |
| KV10130 | ΔrscS::FRT | Derived from KV9501 | This study |
| KV10154 | IG::rscS-3′ end-FLAG | TT KV8232 with SOE products amplified with primers 2290 and 3138 (gKV10083) and primers 2354 and 1487 (gKV10083) | This study |
| KV10162 | IG::PrscS-rscS-FLAG | TT KV10154 with SOE product amplified with primers 2185 and 85 (gKV10101) | This study |
| KV10163 | ΔsypE::FRT | Derived from KV10020 | This study |
| KV10165 | ΔrscS IG::PrscS-rscS-FLAG-FRT-erm | TT KV3378 with gKV10162 | This study |
| KV10166 | ΔrscS::FRT IG::PrscS-rscS-FLAG | TT KV10130 with gKV10162 | This study |
| KV10181 | ΔrscS::FRT ΔsypE::FRT-Erm | NT10130 with gKV10020 | This study |
| KV10185 | ΔhnoX::FRT-Spec | TT ES114 with SOE product amplified with primers 2155 and 2156 (ES114), primers 2089 and 2090 (pKV521), and primers 2157 and 2158 (ES114) | This study |
| KV10186 | ΔsypF::FRT-Spec | TT ES114 with SOE product amplified with primers 1194 and 1160 (ES114), primers 2089 and 2090 (pKV521), and primers 2297 and 271 (ES114) | This study |
| KV10226 | ΔhnoX::FRT-Spec ΔsypF attTn7::sypF-HPT-FLAG | Derived from KV7226 using KV10185 | This study |
| KV10255 | IG::PnrdR-sypF-FLAG | TT KV8232 with SOE product amplified with primers 2290 and 4067 (gKV10019) and primers 2354 and 1487 (pKV503) | This study |
| KV10255 | IG::PnrdR-sypF-FLAG | TT KV8232 with SOE product amplified with primers 2290 and 4067 (gKV10019) and primers 2354 and 1487 (pKV503) | This study |
| KV10298 | IG::PrscS-rscS/sypF chimera2 | TT KV8232 with SOE product amplified with primers 2290 and 3350 (KV10162) and primers 3351 and 1487 (KV10255) | This study |
| KV10299 | IG::PrscS-rscS-Δperiplasmic loop | TT KV8232 with SOE product amplified with primers 2290 and 4111 (KV10165) and primers 4112 and 1487 (KV10165) | This study |
| KV10301 | ΔsypF::FRT IG::PrscS-rscS/sypF chimera | TT KV5367 with gKV10298 | This study |
| KV10302 | ΔrscS::FRT IG::PrscS-rscS/sypF chimera | TT 10130 with gKV10298 | This study |
| KV10303 | ΔrscS::FRT IG::PrscS-rscS-Δperiplasmic loop | TT KV10130 with gKV10299 | This study |
| KV10304 | ΔhahK ΔsypQ::FRT-Cm IG::PsypA lacZ attTn7::Erm | TT KV8079 with gKV7964 | This study |
| KV10305 | ΔsypF::FRT-Spec ΔrscS::FRT | TT10130 with gKV10186 | This study |
| KV10307 | ΔsypG ΔsypQ::FRT-Cm IG::PsypA lacZ attTn7::Erm | TT KV8079 with PCR-amplified DNA from KV9191 | This study |
| KV10312 | ΔsypF::FRT-Spec ΔrscS::FRT IG::PrscS-rscS/sypF chimera2 | TT 10305 with gKV10298 | This study |
| KV10317 | ΔsypF ΔsypQ::FRT-Cm IG::PsypA lacZ attTn7::Erm | TT KV8079 with PCR-amplified DNA from KV10186 | This study |
| KV10351 | ΔsypF::FRT ΔrscS::FRT IG::PrscS-rscS/sypF chimera | Derived from KV10312 | This study |
| KV10355 | ΔsypF::FRT IG::PrscS-rscS/sypF chimera | Derived from KV10301 | This study |
| KV10388 | KB2B1 ΔrscS::FRT-Spec IG::PrscS-rscS-FLAG (ES114) | TT KV10409 with gKV9501 | This study |
| KV10389 | KB2B1 IG::PnrdR-sypF-FLAG (ES114) | TT KB2B1with gKV10255 | This study |
| KV10390 | PrscS-rscS/sypF chimera (rscS native site) IG (rscS-glpK)::FRT-Erm | TT ES114 with SOE product amplified with primers 40 and 3141 (gKV10302) and primers 2354 and 2921 (gKV9782) | This study |
| KV10391 | attTn7::sypG-D53E-FLAG ΔrscS::FRT-Spec | TT KV6527 with gKV9501 | This study |
| KV10397 | ΔsypF::FRT IG::PrscS-rscS/sypF chimera + PrscS-rscS/sypF chimera (rscS native site) IG (rscS-glpK)::FRT-Erm | TT KV10351 with gKV10390 | This study |
| KV10409 | KB2B1 IG::PrscS-rscS-FLAG (ES114) | TT KB2B1 with gKV10162 | This study |
| KV10439 | ΔrscS::FRT IG::PrscS-rscS-H412Q-FLAG | TT KV10130 with SOE product amplified with primers 2290 and 4239 (gKV10165) and primers 4238 and 1487 (gKV10165) | This study |
| KV10440 | ΔrscS::FRT ΔsypE::FRT | Derived from KV10181 | This study |
| KV10442 | attTn7::sypG-D53E-FLAG ΔrscS::FRT ΔsypE::FRT | TT KV10440 with gKV6527 | This study |
| KV10444 | attTn7::sypG-D53E-FLAG ΔsypE::FRT | TT KV10163 with gKV6527 | This study |
a
HA, HA epitope tagged; flag, FLAG epitope tagged; IG, intergenic between yeiR and glmS (adjacent to the Tn7 site) unless otherwise noted; FRT, the antibiotic cassette was resolved using Flp recombinase, leaving a single FRT sequence; TT, TfoX-mediated transformation of a tfoX-overexpressing version of the indicated strain with the indicated genomic DNA (gDNA) or with a PCR-SOE product generated using the indicated primers and templates.
b
The rscS/sypF chimera substituted the HPT domain of rscS with that of sypF. It was positioned either at the nonnative site between yeiR and glmS (“IG”) under the control of PrscS or at the native rscS location in the ΔsypF ΔrscS background (with the chimera repairing the rscS deletion).