TABLE 1.
Bacterial strains and plasmids used in this work
| Strain, phage, or plasmid | Descriptiona | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| S17.1 | RP4 2-Tc::Mu-Km::Tn7 pro res mod+ | 42 |
| S17.1 (λpir) | λpir recA thi pro hsdR-M+ RP4 2-Tc::Mu-Km::Tn7TpRSMR | 6 |
| SURE | sbcC recB recJΔ(mcrCB-hsdSMR-mrr) endA1 gyrA96 Tcr Kmr | 19 |
| P. fluorescens | ||
| NCIMB 10525 | Nonmucoid P. fluorescens wild type | NCIMB |
| Pf201 | algG+, mucoid P. fluorescens mutant derived from NCIMB 10525 | P. Karunakaran, unpublished data |
| Pf2012 | Mannuronan-producing mutant, algG D361N | This work |
| Pf2013 | Mannuronan-producing mutant, algG G430D | This work |
| Pf20117 | Mannuronan-producing mutant, algG R408C | This work |
| Pf20118 | Mannuronan-producing mutant, algG R408C | This work |
| Pf20137 | Mannuronan-producing mutant, algG S337F | This work |
| Pf20138 | Mannuronan-producing mutant, algG S337F | This work |
| Pf201ΔalgG | algG in-frame deletion mutant | This work |
| Pf20118::TnKB10 | Derivative of Pf20118 with transposon from pKB10 | This work |
| Pf201ΔalgG::TnKB10 | Derivative of Pf201ΔalgG with transposon from pKB10 | This work |
| Pf201ΔalgG::TnCNB111 | Derivative of Pf201ΔalgG with transposon from pCNB111 | This work |
| Phages and plasmids | ||
| λ DashII | λ cloning vector | Stratagene |
| Pfλ1 | λ DashII with an ∼15 kb insert of Sau3AI partially digested genomic DNA from P. fluorescens NCIMB10525 encoding alg′EGXLIJFA | M. Gimmestad, unpublished data |
| pGEM5 | ColE1; Apr | Promega |
| pGEM11 | ColE1; Apr | Promega |
| pCVD442 | oriR6K; Apr | 7 |
| pJB3Tc20 | RK2-based vector; Apr Tcr | 2 |
| pCNB111 | oriR6K mobRP4, pUT/mini-Tn5 xylS/Pm; Apr Kmr | 49 |
| pCNB111luc | oriR6K mobRP4, pUT/mini-Tn5 xylS/Pm, luc; Apr Kmr | 49 |
| pJB3Tc20trfA | Derivative of pJB3Tc20 from which a 1.0-kb BsaAI-NdeI DNA fragment encoding TrfA was deleted | This work |
| Litmus28 | ColE1; Apr | New England Biolabs |
| pHE55 | Derivative of pJB3Tc20trfA in which a 2.6-kb PstI-XbaI DNA fragment from pCVD442 encoding SacB from B. subtilis was inserted | This work |
| pJB1002 | RK2-based vector encoding a TrfA-LacZ-fusion protein | 25 |
| pMG23 | Litmus28 in which a 1.8-kb PCR-amplified BglII-PstI DNA fragment containing algG and 135 bp of algX was inserted; the primers PfalgG3r and PfalgG4f were used for amplification | This work |
| pMG26 | pGEM11 containing a 4.6-kb SalI DNA fragment from Pfλ1 | M. Gimmestad, unpublished data |
| pMG31 | Derivative of pHE55 in which an 1.8-kb BglII-XbaI DNA fragment encoding algG from pMG23 was inserted | This work |
| pMG47 | XbaI/PstI-restricted derivative of pHE55 in which a 4.1-kb NheI-PstI DNA fragment from pJB1002 encoding the TrfA-LacZ fusion protein was inserted | This work |
| pMG48 | pMG47 with a 0.36-kb SphI-SapI DNA fragment containing the polylinker of pGEM5 | This work |
| pMG51 | Derivative of pMG26 in which a SmaI site was introduced at nucleotide position 368 in algG by using the primers algG-SmaI-1 and algG-SmaI-2 | This work |
| pMG52 | Derivative of pMG51 from which a 0.6-kb SmaI DNA fragment was deleted, creating an in-frame deletion in algG | This work |
| pMG53 | Derivative of NsiI-NcoI-restricted pMG48 in which a 2.1-kb PstI-BspHI DNA fragment from pMG52 was inserted | This work |
| pKB4 | Derivative of pMG26 from which a 3.0-kb BlpI-XhoI DNA fragment was deleted; | This work |
| pKB10 | Derivative of pCNB111luc in which luc was replaced with a 1.7-kb NdeI-NotI-restricted PCR fragment containing algG pKB4 was used as PCR template and PfalgG-NdeI-2 and M13/pUC reverse were used as primers | This work |
a
Tcr, Kmr, and Apr, tetracycline, kanamycin, and ampicillin resistance, respectively.