TABLE 1.
Bacterial strains and plasmids used in this study
| Bacterial strain or plasmid | Relevant characteristic(s)a | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| BL21 | F−ompT hsdSB(rB− mB−) gal dcm | Novagen |
| DH5α | supE44 thi-1 recA1 hsdR17 endA1 gyrA (Nalr) Δ(lacIZYA-argF) U169 deoR [φ80dlac Δ(lacZ)M15] | 62 |
| S17-1 | recA pro thi hsdR RP4-2-Tc::Mu-Km::Tn7 Trar+ Tpr Smr | 66 |
| XL1-Blue | recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 [F′ laclZM15 Tn10 (Tetr)] | 10 |
| P. fluorescens | ||
| KU-7 | Wild type, 2-NBA− 4-HBA+ | 27 |
| KUM-9 | orf16::Tn5-31 Tp 2-NBA− 4-HBA+ | This study |
| KUM-19 | nbaA::Tn5-31 Tp 2-NBA− 4-HBA+ | This study |
| KUM-33 | nbaB::Tn5-31 Tp 2-NBA− 4-HBA+ | This study |
| KU-ΔY | nbaY deletion mutant | This study |
| Plasmids | ||
| pKN31 | Tn5-31 Tp transposon delivery plasmid; mob RP4 Kmr Tpr | 1 |
| pK18mobsacB | Negative selection vector; Kmr | 63 |
| pREP1 | Expression vector with T7 promoter, p15A origin; Cmr | 57 |
| pSD80 | Expression vector with tac promoter, ColE1 origin,r Apr | 68 |
| pT7-5 | Expression vector with T7 promoter, ColE1 origin,r Apr | 70 |
| pUC19 | Cloning vector with lac promoter, ColE1 origin,r Apr | 74 |
| pNBA3 | 9.3-kb HindIII fragment from P. fluorescens strain KU-7 in pUC19; Apr | 49 |
| pNBA5 | 6.2-kb EcoRI fragment from P. fluorescens strain KUM-19 in pUC19; Apr Tpr | This study |
| pNBA6 | 3.8-kb BamHI fragment from P. fluorescens strain KUM-19 in pUC19; Apr Tpr | This study |
| pSDnbaA | EcoRI*-PstI* fragment containing nbaA in pSD80 | This study |
| pSDnbaA-N40A | Replacement of Asn40 by Ala in NbaA in pSDnbaA | This study |
| pSDnbaA-H63A | Replacement of His63 by Ala in NbaA in pSDnbaA | This study |
| pSDnbaA-H69A | Replacement of His69 by Ala in NbaA in pSDnbaA | This study |
| pSDnbaA-D76A | Replacement of Asp76 by Ala in NbaA in pSDnbaA | This study |
| pSDnbaA-H110A | Replacement of His110 by Ala in NbaA in pSDnbaA | This study |
| pSDnbaA-E113A | Replacement of Gln113 by Ala in NbaA in pSDnbaA | This study |
| pSDnbaB | EcoRI*-PstI* fragment containing nbaB in pSD80 | This study |
| pSDnbaAnbaB | pSDnbaA plus ribosome binding site at HindIII and NheI plus nbaB cloned at AscI and NheI | This study |
| pNbaΔY | nbaY deletion cassette in pK18mobsacB | This study |
a
EcoRI* and PstI* are restriction endonucleases introduced by PCR design. 2-NBA+, growth on 2-NBA: 4-HBA+, growth on 4-hydroxybenzoate; 2-NBA−, no growth on 2-NBA.