Table 3.
Plasmids used in this study.
| Plasmids Provided by Others | |
|---|---|
| Designation | Relevant Features or Description |
| pABB19 | oriMB1, Apr, transcriptional terminator Tpro/Tlyz P1 [51] |
| pABB28.2 | pET28a with his-tag replaced by flag-tag [52] |
| pAKB2.55 | pGBT30 with kfrC without a stop codon (IBB) a |
| pAKB2.70 | pGBT30 with kfrCRA3-yfp (IBB) a |
| pAKB7.5 | oriMB1, Kmr, parS-oriTRA3 (RA3 coordinates 9397–9854 nt) [24] |
| pAKB16.50 | pLKB4 cyaT18-korBRA3 [17] |
| pAKB16.50N | pKGB4 korBRA3-cyaT18 [17] |
| pAMB8 | pBBR1MCS-3 modified in tetM to remove EcoRI site (IBB) a |
| pBBR1MCS | IncA/C, Cmr, BHR cloning vector [53] |
| pBBR1MCS-2 | IncA/C, Kmr BHR cloning vector [54] |
| pBGS18 | oriMB1, Kmr, cloning vector [55] |
| pESB5.58 | pGBT30 with tacp-kfrA [18] |
| pET28a | oriMB1, Kmr, T7p, lacO, His6-tag, T7 tag (Novagen) |
| pET28mod | pET28a derivative, T7 tag removed [56] |
| pGBT30 | oriVMB1, Apr, lacIq, tacp expression vector [57] |
| pJSB8.5.2 | pLKB4 cyaT18-virD4 (IBB) a |
| pJSB9.5.1 | pKGB4 virD4-cyaT18 (IBB) a |
| pJSB10.5.2 | pLKB2 cyaT25-virD4 (IBB) a |
| pJSB11.5.1 | pKGB5 virD4-cyaT25 (IBB) a |
| pKAB20 | pUC19 derivative with flag-mcsb-his6; allows in-frame attachment of flag to 5′ and/or his6 to the 3′ of a gene [58] |
| pKAB28 | pET28mod with deletion of his6-tag and EcoRI site adjacent to RBS [57] |
| pKAB28.7 | pET28mod derivative with his6- mcsb [58] |
| pKD13 | template plasmid for gene disruption [30] |
| pKD46 | oriR101, araBp-gam-bet-exo, repA101(ts), Apr, lambda Red recombinase expression plasmid [30] |
| pKGB4 | oriColE1, pUT18 with modified mcs, lacp- mcsb -cyaT18, Apr (IBB) a |
| pKGB5 | orip15, pKNT25 with modified mcs, lacp- mcsb -cyaT25, Kmr (IBB) a |
| pKT25-zip | pKT25 derivative encoding CyaT25 in translational fusion with leucine zipper of GCN4 [34] |
| pLKB2 | orip15, pKT25 with modified mcs, lacp-cyaT25- mcsb, Kmr [59] |
| pLKB4 | oriColE1, pUT18C with modified mcs, lacp-cyaT18- mcsb, Apr [59] |
| pMRA1.3 | pLKB4 with cyaT18-kfrCR751 (IBB) a |
| pMRB2.3 | pKGB4 with kfrCR751-cyaT18 (IBB) a |
| pMRB3.3 | pLKB2 with cyaT25-kfrCR751 (IBB) a |
| pMRB4.3 | pKGB5 with kfrCR751-cyaT25 (IBB) a |
| pOMB3.104 | pUC18 derivative with parS P1 prophage (IBB) a |
| pOMB4.13 | pLKB4 with cyaAT18-kfrA [18] |
| pOMB4.15 | pLKB4 with cyaAT18-kfrC [18] |
| pOMB5.13 | pLKB2 with cyaAT25-kfA [18] |
| pOMB5.15 | pLKB2 with cyaAT25-kfrC [18] |
| pOMB6.14 | pKGB4 with kfrA-cyaAT18 [18] |
| pOMB6.16.1 | pKGB4 with kfrC-cyaAT18 [18] |
| pOMB7.14 | pKGB5 with kfrA-cyaAT25 [18] |
| pOMB7.16.1 | pKGB5 with kfrC-cyaAT25 [18] |
| pOMB9.80 | pGBT30 with kfrARA3-cfp (IBB) a |
| pUC18 | oriMB1, Apr, cloning vector [60] |
| pUT18C-zip | pUT18C derivative encoding CyaT18 in translational fusion with leucine zipper of GCN4 [34] |
| R751TcR | IncPβ (IncP-1β) c, Tcr-derivative of R751 [38] |
| RA3 | IncU (IncP-6) c, Cmr, Smr, Sur (F. Hayes) |
| RK2 | IncPα (IncP-1α) c, Apr, Kmr, Tcr (C.M. Thomas) |
| Plasmids Constructed during This Work | |
| Designation | Relevant Features or Description |
| pESB5.88 | pGBT30 with tacp-kfrC; annealed oligonucleotides 28 and 29 inserted between Xba-SalI of pAKB2.55 |
| pESB5.90 | pGBT30 with tacp-kfrC without a stop codon; annealed oligonucleotides 6 and 7 inserted between Xba-SalI of pAKB2.55 |
| pESB10 | pBBR1MCS-2 lacIq tacp with transcriptional terminator T1/T2rrnB; PCR product obtained with primers 36 and 37 on E. coli genomic DNA inserted as XhoI-KpnI fragment between SalI-KpnI sites |
| pESB11 | pOMB12.0 derivative with transcriptional terminator T1/T2rrnB; PCR fragment obtained with primers 36 and 37 on E. coli genomic DNA inserted between XhoI-KpnI sites |
| pESB11.58 | pESB11 with tacp-kfrA; EcoRI-SalI fragment from pESB5.58 inserted between EcoRI-XhoI sites |
| pESB15 | pET28a with annealed oligonucleotides 30 and 31 inserted between NcoI and BamHI sites |
| pESB15.90 | pESB15 with kfrC-his6; EcoRI-HindIII fragment from pESB5.90 |
| pJSB1.4 | pBGS18 with the mobCp-mobC-nic; PCR fragment obtained with primers 26 and 5 on RA3 template inserted between EcoR-SalI sites (RA3 coordinates 9437–11355 nt) |
| pJSB1.5.2 | pBGS18 with virD4; PCR fragment obtained with primers 44 and 45 on RA3 template cloned between the BamHI-KpnI sites (RA3 coordinates 18230–16305 nt) |
| pJSB1.8 | pBGS18 with TraRA3; pJSB1.4 with SmaI-SalI fragment of RA3 plasmid (RA3 coordinates 10733–22925 nt) |
| pJSB1.24 | pBGS18 with TraRA3-korCp-korC; PCR fragment korCp-korC obtained with primers 2 and 3 (RA3 coordinates 3093–3705) inserted into pJSB1.8 |
| pJSB8.1 | pLKB4 with cyaT18-mobC; PCR fragment obtained with primers 22 and 23, cloned between the EcoRI-HincII sites (RA3 coordinates 9837–10455 nt) |
| pJSB8.3 | pLKB4 with cyaT18-nic; PCR fragment obtained with primers 25 and 26 cloned between the EcoRI-HincII sites (RA3 coordinates 10360–11355 nt) |
| pJSB8.5.2 | pLKB4 with cyaT18-virD4; fragment BamHI-KpnI from pJSB1.5.2 cloned into pLKB4 |
| pJSB8.35 | pLKB4 with cyaT18-mobC-oriTRA3; SmaI-HincII fragment of pAKB7.5 carrying parS-oriT cloned into PvuII site of pJSB8.1 |
| pJSB9.1.1 | pKGB4 with mobC-cyaT18; PCR fragment obtained with primers 22 and 24 cloned between EcoRI-SacI sites, (RA3 coordinates 9837–10364 nt) |
| pJSB9.5.1 | pKGB4 with virD4-cyaT18; PCR fragment obtained with primers 44 and 46 cloned between BamHI-SacI sites (RA3 coordinates 18230–16308 nt) |
| pJSB10.1 | pLKB2 with cyaT25-mobC; PCR fragment EcoRI-HincII obtained with primers 22 and 23, cloned between the EcoRI-SmaI sites (RA3 coordinates 9837–10455 nt) |
| pJSB10.3 | pLKB2 with cyaT25-nic; PCR fragment EcoRI-HincII obtained with primers 25 and 26 cloned between EcoRI-SmaI sites (RA3 coordinates10360–11355 nt) |
| pJSB10.5.2 | pLKB2 with cyaT25-virD4; PCR fragment obtained with primers 44 and 45 cloned between BamHI-KpnI sites (RA3 coordinates18230–16305 nt) |
| pJSB11.1 | pKGB5 with mobC-cyaT25; PCR fragment obtained with primers 22 and 24 cloned between EcoRI-SacI sites (RA3 coordinates 9837–10364 nt) |
| pJSB11.3 | pKGB5 with nic-cyaT25; PCR fragment obtained with primers 25 and 27 cloned between EcoRI-SacI sites (RA3 coordinates 10360–11352 nt) |
| pJSB11.5.1 | pKGB5 with virD4-cyaT25; PCR fragment obtained with primers 44 and 46 cloned between BamHI-SacI sites (RA3 coordinates 18230–16308 nt) |
| pOMB1.17 | pBGS18 with kfrC1–249; PCR product amplified on RA3 template with primers 8 and 9 inserted between EcoRI-SalI sites (RA3 coordinates: 3692–4438) |
| pOMB1.18 | pBGS18 with kfrC244–355; PCR product amplified on RA3 template with primers 10 and 11 inserted between EcoRI-SalI sites (RA3 coordinates: 4421–4756) |
| pOMB1.42 | pBGS18 with virD4434–641; EcoRI-BamHI fragment from pOMB4.42 |
| pOMB1.51 | pBGS18 with virD4434–641 kfrC; PCR product amplified on RA3 template with primers 14 and 18 inserted as BglII-SalI fragment between BamHI-SalI sites of pOMB1.42 (RA3 coordinates: 3686–4756) |
| pOMB1.74 | pBGS18 virD4-cfp; BamHI-HindIII fragment from pOMB9.80 with overhangs filled in using Klenow fragment of PolI inserted within EcoICRI site of pJSB1.5.2 |
| pOMB2.0 | pKAB20 derivative with Ecl136II restriction site inserted between MunI and HindIII sites (annealed oligonucleotides 33 and 34) |
| pOMB2.0.28 | pUC19 with kfrC244–355-his6; EcoRI-SmaI fragment from pOMB1.18 inserted in EcoRI-Ecl136II sites of pOMB2.0 |
| pOMB2.49 | pUC19 with flag-virD4; PCR product amplified on RA3 template with primers 4 and 49 inserted between MunI-HindIII sites of pKAB20 (RA3 coordinates: 18230–16305) |
| pOMB2.50 | pUC19 with flag-vird4434–641; EcoRI-SalI fragment from pOMB1.42 inserted between MunI-SalI sites of pKAB20 |
| pOMB2.52 | pUC19 with flag-virD4434–641 kfrC-his6; EcoRI-SalI fragment from pOMB1.51 inserted between MunI-XhoI sites of pKAB20 |
| pOMB2.74 | pUC19 virD4-cfp; PCR product amplified on pOMB1.74 template with primers 1 and 49 inserted as MunI-SmaI sites of pOMB2.0 |
| pOMB4.0 | pLKB4 derivative with I-SceI restriction site inserted into KpnI site (annealed oligonucleotides 20 and 21) |
| pOMB4.17 | pLKB4 with cyaT18-kfrC1–249; EcoRI-SmaI fragment from pOMB1.17 |
| pOMB4.18 | pLKB4 with cyaT18-kfrC244–355; EcoRI-SmaI fragment from pOMB1.18 |
| pOMB4.34 | pLKB4 with cyaT18-traGR751; PCR product amplified on R751 template with primers 38 and 39 inserted as EcoRI-KpnI fragment (R751 coordinates: 48800–46887) |
| pOMB4.36 | pLKB4 with cyaT18-kfrCRK2; PCR product amplified on RK2 template with primers 15 and 16 inserted as EcoRI-KpnI fragment (RK2 coordinates: 54424–53079) |
| pOMB4.38 | pLKB4 with cyaT18-traGRK2; PCR product amplified on RK2 template with primers 40 and 41 inserted as EcoRI-KpnI fragment (RK2 coordinates: 48495–46588) |
| pOMB4.42 | pLKB4 with cyaT18-virD4434–641; PCR product amplified on RA3 template with primers 47 and 48 inserted between EcoRI-BamHI sites (RA3 coordinates: 16931–16305) |
| pOMB5.17 | pLKB2 with cyaT25-kfrC1–249; EcoRI-SmaI fragment from pOMB1.17 |
| pOMB5.18 | pLKB2 with cyaT25-kfrC244–355; EcoRI-SmaI fragment from pOMB1.18 |
| pOMB5.34 | pLKB2 with cyaT25-traGR751; EcoRI-KpnI fragment from pOMB4.34 |
| pOMB5.36 | pLKB2 with cyaT25-kfrCRK2; EcoRI-KpnI fragment from pOMB4.36 |
| pOMB5.38 | pLKB2 with cyaT25-traGRK2; EcoRI-KpnI fragment from pOMB4.38 |
| pOMB6.17 | pKGB4 with kfrC1–249-cyaT18; EcoRI-SmaI fragment from pOMB1.17 |
| pOMB6.18 | pKGB4 with kfrC244–355-cyaT18; EcoRI-SmaI fragment from pOMB1.18 |
| pOMB6.35 | pKGB4 with traGR751-cyaT18; PCR product amplified on R751 template with primers 38 and 43 inserted as EcoRI-SmaI fragment (R751 coordinates: 48800–46890) |
| pOMB6.37 | pKGB4 with kfrCRK2-cyaT18; PCR product amplified on RK2 template with primers 15 and 17 inserted as EcoRI-SmaI fragment (RK2 coordinates: 54424–53082) |
| pOMB6.39 | pKGB4 with traGRK2-cyaT18; PCR product amplified on RK2 template with primers 40 and 42 inserted as EcoRI-SmaI fragment (RK2 coordinates: 48495–46591) |
| pOMB7.17 | pKGB5 with kfrC1–249-cyaT25; EcoRI-SmaI fragment from pOMB1.17 |
| pOMB7.18 | pKGB5 with kfrC244–355-cyaT25; EcoRI-SmaI fragment from pOMB1.18 |
| pOMB7.35 | pKGB5 with traGR751-cyaT25; EcoRI-SmaI fragment from pOMB6.35 |
| pOMB7.37 | pKGB5 with kfrCRK2-cyaT25; EcoRI-SmaI fragment from pOMB6.37 |
| pOMB7.39 | pKGB5 with traGRK2-cyaT25; EcoRI-SmaI fragment from pOMB6.39 |
| pOMB8.28 | pET28mod with kfrC244–355-his6; pKAB28 derivative with EcoRI-SalI fragment from pOMB2.0.28 |
| pOMB8.49 | pET28mod with flag-virD4; MunI-HindIII fragment from pOMB2.49 inserted between EcoRI-HindIII sites of pKAB28 |
| pOMB8.50 | pET28mod with flag-virD4434–641; pKAB28 derivative with EcoRI-SalI fragment from pOMB2.50 |
| pOMB8.52 | pET28mod with flag-virD4434–641 kfrC-his6; pKAB28 derivative with EcoRI-SalI fragment from pOMB2.52 |
| pOMB9.18 | pGBT30 with tacp-kfrC244–355; EcoRI-SalI fragment from pOMB1.18 |
| pOMB9.29 | pGBT30 with tacp-kfrC-his6; PCR product amplified on the pESB15.90 template with primers 19 and 35 inserted between XbaI-SalI sites of pESB5.88 |
| pOMB9.31 | pGBT30 with tacp-kfrC*; two-stage PCR was used for KfrC site-directed mutagenesis, described in detail in Metods, PCR final product was inserted between XbaI-SalI sites |
| pOMB12.0 | pOMB12.30 derivative with transcriptional terminator Tpro/Tlyz P1; PCR product amplified on pABB19 as a template with primers 50 and 51 inserted as EcoRI-SalI fragment between EcoRI-XhoI sites |
| pOMB12.15 | pESB11 with tacp-kfrC; EcoRI-SalI fragment from pESB5.88 inserted between EcoRI-XhoI sites |
| pOMB12.30 | pBBR1MCS-3 lacIq tacp; pAMB8 derivative with EcoRI-PstI fragment from pGBT30 |
| pOMB12.74 | pBBR1MCS-2 virD4-cfp; pESB10 derivative with MunI-SmaI fragment from pOMB2.74 inserted between EcoRI-SmaI sites |
| RA3ΔincC | incC gene replaced by Kmr cassette amplified on pKD13 template with primers 52 and 53 (coordinates of deletion: 6356–7080) |
| RA3Δnic | nic gene replaced by Kmr cassette amplified on pKD13 template with primers 54 and 55 (coordinates of deletion: 10380–11352) |
| RA3ΔvirD4 | virD4 replaced by Kmr cassette amplified on pKD13 template with primers 56 and 57 (coordinates of deletion: 18195–16314) |
| RA3ΔparS | parS site replaced by Kmr cassette amplified on pKD13 template with primers 58 and 59 (coordinates of deletion: 9707–9722) |
| RA3ΔoriT | oriT site replaced by Kmr cassette amplified on pKD13 template with primers 60 and 61 (coordinates of deletion: 9747–9756) |
| RA3parS▼oriT | Kmr cassette amplified on pKD13 template with primers 59 and 62 and inserted between parS and oriT sites (coordinates of insertion: 9722/9723) |
| RA3ΔkfrA | kfrA replaced by Kmr cassette amplified on pKD13 template with primers 63 and 64 (coordinates of deletion: 4892–5935) |
| RA3ΔkfrC | kfrC replaced by Kmr cassette amplified on pKD13 template with primers 65 and 66 (coordinates of deletion: 3695–4738) |
| RA3Δ(kfrC-A) | kfrC-kfrA replaced by Kmr cassette amplified on pKD13 template with primers 65 and 64 (coordinates of deletion: 3695–5935) |
| RA3# | parSP1-Kmr cassette inserted within integron at position 38,663 of RA3 genome |
a—Institute of Biochemistry & Biophysics collection; b—mcs, multiple-cloning site modified; c—in brackets plasmid incompatibility groups in Pseudomonas spp.