Table 1.
Bacterial strains, plasmids, and oligonucleotides used in the study.
| E. coli | ||
| DH5α | F−endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 Δ(lacZYA-argF) U169 deoR λ(φ80dlacZΔM15) | TakaRa |
| S17-1 λpir | pro thi hsdR+ Tpr Smr; chromosome::RP4-2 Tc::Mu-Kan::Tn7/λpir | Simon et al., 1983 |
| P. aeruginosa | ||
| PAO1 | Wild-type | Stover et al., 2000 |
| PAO1 ΔrpoN | PAO1 in-frame deletion of rpoN | Viducic et al., 2016 |
| PAO1 ΔrpoNΔrpoS | PAO1 in-frame deletion of rpoN and rpoS | This study |
| PAO1 ΔrpoS | PAO1 in-frame deletion of rpoS | Viducic et al., 2017 |
| PAO1 ΔrpoN/rpoN+ | ΔrpoN mutant carrying a wild-type copy of rpoN on pMMB67EH | This study |
| Plasmids | ||
| pEX18Gm | Broad-host-range gene replacement vector; sacB, Gmr | Hoang et al., 1998 |
| pEX18Gm-ΔrpoN | rpoN deletion suicide vector | Viducic et al., 2016 |
| pEX18Gm-ΔrpoS | rpoS deletion suicide vector | Viducic et al., 2017 |
| pMMB67EH | IncQ broad-host-range cloning vector, Apr | Fürste et al., 1986 |
| pMMB67EH-rpoN | pMMB67EH containing a functional rpoN gene in the opposite orientation to the tac promoter, Apr | This study |
| Primers for mutant construction and complementation: | ||
| rpoN-up-F | ATAGAATTCCGATCTCGGTCGGCGACATC | Viducic et al., 2016 |
| rpoN-up-R | ATAGGATCCCTGGAGGTCCAGGGTGGATAG | Viducic et al., 2016 |
| rpoN-down-F | ATAGGATCCGGCATAGCCCCTTCGAGCGAG | Viducic et al., 2016 |
| rpoN-down-R | ATAAAGCTTCTCCGGCAGCTCCCTGGCTA | Viducic et al., 2016 |
| rpoS-up-F | CATTCAGGTCGGTCAAGCTATCCA | Viducic et al., 2017 |
| rpoS-up-R | TCCGTCACTGTGCCATGTCGTTATCCCTTG | Viducic et al., 2017 |
| rpoS-down-F | CGACATGGCACAGTGACGGAAAACCTTAGA | Viducic et al., 2017 |
| rpoS-down-R | GGAAGTCTGGCCGAACATCACGA | Viducic et al., 2017 |
| rpoN-HindIII-F | CCCAAGCTTGGGAAGTCTACCTGGGGCACGAGT | This study |
| rpoN-EcoRI-R | CCGGAATTCCGGTCCTTGTTGCCCGTGTGTAAGT | This study |
| Primers for qRT-PCR: | ||
| rsmA-F | TGGGTGTCAAAGGGAACCA | This study |
| rsmA-R | TGGTAAATTTCCTCCCGGTGTA | This study |
| relA-F | CCCCAAGGAAAACGGCTATC | This study |
| relA-R | TGGAGTGGGTACGGATCTGTACT | This study |
| rpoS-F | CACTTCCTTCTCTCCAAACAACA | Viducic et al., 2016 |
| rpoS-R | AGCTGCGTTGCGTCCAA | Viducic et al., 2016 |
| omlA-F | CGAACTATCAACCAGCTGGTG | Viducic et al., 2016 |
| omlA-R | GCTGTGCTCTTGCAGGTTGTG | Viducic et al., 2016 |
aTpr, Smr, Apr, and Gmr, resistance to trimethoprim, streptomycin, ampicillin, and gentamicin, respectively. Introduced restriction sites are underlined, and the sequences introduced for overlap extension PCR are in italics.