TABLE 1.
Plasmids and strains used in this work
| Strain or plasmid | Description | Reference or source |
|---|---|---|
| Strains | ||
| E. coli | ||
| S17.1 | RP4 2-Tc::Mu-Km::Tn7 pro res mod+ | 33 |
| S17.1(λpir) | λpirn lysogen of strain S17.1 | 7 |
| P. fluorescens | ||
| NCIMB10525 | Nonmucoid P. fluorescens wild type | NCIMB |
| Pf201 | Mucoid P. fluorescens mutant derived from NCIMB 10525 | 19 |
| Pf20118 | Mannuronan-producing derivative of Pf201, algG(R408C) | 18 |
| Pf20118ΔalgL | Derivative of Pf20118 containing ΔalgL from pMG69 | This work |
| Pf20118algLH203R | Derivative of Pf20118 with the algL(H203R) point mutation from pMG70 | This work |
| Pf20118algLH203R::TnKB16 | Derivative of Pf20118algLH203R with transposon TnKB16 from pKB16 | This work |
| Pf201ΔalgC | Pf201 derivative with the ΔalgC deletion from pKB22 | This work |
| Pf201ΔalgC::TnKB60 | Derivative of Pf201ΔalgC with transposon TnKB60 from pKB60 | This work |
| Pf201ΔalgCΔalgL | Pf201ΔalgC with the ΔalgL from pMG69 | This work |
| Pf201ΔalgCΔalgL::TnKB60 | Derivative of Pf201ΔalgCΔalgL with the TnKB60 transposon from pKB60 | This work |
| Pf201ΔalgCΔalgL::TnKB60::TnKB62 | Derivative of Pf201ΔalgCΔalgL::TnKB60 with the TnKB62 transposon from pKB62 | This work |
| Pf201ΔalgCalgLH203A | Pf201ΔalgC with the algL(H203A) point mutation from pMG92 | This work |
| Pf201ΔalgCalgLH203A::TnKB60 | Pf201ΔalgCalgLH203A with the TnKB60 transposon from pKB60 | This work |
| Plasmids | ||
| pLitmus28 | ColE1 replicon; Apr. | New England Biolabs Inc. |
| pUC7Tc | Derivative of pUC7 in which Tcr gene from RK2 is inserted | 3 |
| pLitmus28Tc | Litmus28(BamHI) with an insertion of a 2.3-kb fragment (BamHI) containing tetA and tetR from pUC7Tc; Apr Tcr. | This work |
| pCNB111luc | oriR6K mobRP4, pUT/mini-Tn5 xylS/Pm luc Apr Kmr. | 40 |
| pMG26 | High-copy-number cloning vector encoding alg′ GXLI from P. fluorescens NCIMB 10525; Apr. | 18 |
| pMG48 | RK2-based gene replacement vector; Apr Tcr. | 18 |
| pMG66 | Derivative of pMG26 in which a NruI site was introduced in position 660 of algL. The primer pair algL-NruI-1 and algL-NruI-2 was used for site-specific mutagenesis; Apr. | This work |
| pMG67 | Derivative of pMG26 in which the algL(H203R) mutation was generated by site-specific mutagenesis, using the primer pair algLH203R1 and algLH203R2; Apr. | This work |
| pMG68 | Derivative of pMG66 from which a 0.4-kb NruI fragment was deleted, resulting in an in-frame deletion in algL; Apr. | This work |
| pMG69 | Derivative of NsiI-NotI-restricted pMG48 with insertion of a 2.9-kb fragment from pMG68 (PstI-NotI) encoding ΔalgL; Apr Tcr. | This work |
| pMG70 | Derivative of NsiI-NotI-restricted pMG48 with insertion of a 2.9-kb fragment from pMG67 (PstI-NotI) encoding algL(H203R); Apr, Tcr. | This work |
| pMG91 | Derivative of pMG26 in which the algL(H203A) mutation was generated by site-specific mutagenesis, using the primer pair algLH203A1 and algLH203A2; Apr. | This work |
| pMG92 | Derivative of NsiI-NotI-digested pMG48 with an insertion of a 2.9-kb fragment from pMG91 (PstI-NsiI) encoding algL(H203A); Apr Tcr. | This work |
| pJBphOx-271 | RK2-based expression vector harboring the Pm/xylS-regulated promoter system and the trfA271C copy up mutation; Apr. | 35 |
| pMC2 | Derivative of pKB10 in which algG was replaced with a 1.4-kb PCR-amplified NdeI-NotI fragment containing algC PCR amplified from P. fluorescens NCIMB 10525 total DNA. Primers PfalgCNdeIfor and PfalgCNotlrev were used for amplification; Apr Kmr. | This work |
| pKB10 | Suicide vector encoding a mini-Tn5 transposon with algG under Pm control; Apr Kmr. | 18 |
| pKB16 | Derivative of NdeI-NotI-restricted pCNB111luc in which luc was replaced with a 1.6-kb PCR-amplified fragment (NdeI-NotI) encoding algL. pMG26 was used as PCR template and PfalgLNdeI and M13/pUC reverse primer were used for amplification. Encodes the transposon TnKB16 with algL under the control of Pm; Apr Kmr. | This work |
| pKB20 | Derivative of pJBphOx-271 in which a 1.4-kb EcoRI-XbaI fragment from pMC2 encoding algC was inserted; Apr. | This work |
| pKB21 | Derivative of pKB20 from which a 0.5-kb EcoRI-PmlI fragment was deleted, creating an in-frame deletion in algC; Apr. | This work |
| pKB22 | Derivative of NsiI-NcoI-restricted pMG48 in which a 0.9-kb NsiI-NcoI fragment containing ΔalgC from pKB21 was inserted; Apr Tcr. | This work |
| pKB60 | Derivative of pKD23 in which algL was replaced with a 1.4-kb NdeI-NotI fragment from pMC2 containing algC. Encodes the transposon TnKB60 with algC under the control of PmG5; Apr Kmr. | This work |
| pKB62 | Derivative of pKD23 from which a 1.7-kb NcoI-AvrII fragment encoding the Kmr gene was replaced with a 2.6-kb fragment (AfllII-AvrII) encoding tetA and tetR from Litmus28Tc. Encodes the transposon TnKB62 with algL under the control of PmG5; Apr Tcr. | This work |
| pKD20 | pKB10 (SacI-NotI) with an insertion of a 1-kb PCR fragment (PstI-SacI) encoding Kmr (primers KanSacI and KanPstI) and a 1.8-kb PCR fragment (PstI-NotI) containing the PmG5 promoter and xylS (primers XylSPstI and XylSNotI) both using pHH100-G5 as template; Apr Kmr. | This work |
| pKD23 | Derivative of pKD20(NdeI-NotI) in which a 1.6-kb PCR-amplified fragment (NdeI-NotI) containing algL was inserted. pMG26 was used as PCR template and PfalgLNdeI and M13/pUC reverse primer were used for amplification; Apr Kmr. | This work |
| pHH100-G5 | RK2-based plasmid containing a mutant Pm promoter designated PmG5; Kmr. | 19 |