TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Genotype and/or relevant characteristicsa | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| α-Select (Silver Efficiency) | deoR endA1 recA1 relA1 gyrA96 hsdR17(rK− mK+) supE44 thi-1 Δ(lacZYA-argFV169) ϕ80dlacZΔM15 F− | Bioline |
| BL21(DE3) competent cells | B F− dcm ompT hsdS(rB− mB−) gal λ (DE3) | Agilent |
| D. vulgaris Hildenborough | ||
| ATCC 29579 | Wild type; 5-FUs | ATCC |
| JW710 | WT Δupp; 5-FUr (used as a WT control for D. vulgaris Hildenborough growth kinetics in this study; parent strain for deletions; retains pDV1 present in WT) | 8 |
| JW3311 | JW710 ΔDVU_0916::(npt upp); Kmr; 5-FUs (Δrex marker exchange) | 33 |
| JW9293 | JW710 Δ−150–1 Psat::(npt upp); Kmr; 5-FUs (Psat disruption) | This study |
| JW9312 | JW710; Kms; 5-FUr (sat promoter restored) | This study |
| JW9314 | JW9293 G−147A Psat; Kms; 5-FUr (G −147 A) | This study |
| JW9316 | JW9293 GTA−147–145ACG Psat; Kms; 5-FUr (IR1) | This study |
| JW9318 | JW9293 CAC−136–134TGT Psat; Kms; 5-FUr (IR2) | This study |
| JW9320 | JW9293 GTA−147–145ACG Psat CAC−136–134TGT Psat; Kms; 5-FUr (IR1and2) | This study |
| JW9011 | JW710 ΔDVU_2547::(npt upp); Kmr; 5-FUs (ΔhcpR marker exchange) | 50 |
| GZ0481 | Genome position 2680507::Tn5-RL27; insertion 273 bp from predicted AUG start codon within DVU_2567; Kmr (LysX mutant) | Wall laboratory |
| Plasmids | ||
| pET14b | 6×His tag fusion protein vector with T7 promoter | Novagen |
| pMO719 | pCR8/GW/TOPO containing SRB replicon (pBG1); Spr; source of Spr and pUC ori fragment; for marker exchange suicide plasmid construction | 8 |
| pMO746 | Source of upp in artificial operon with npt and Apr-pUC ori; Pnpt-npt-upp; Kmr; 5-FUs; for marker exchange suicide plasmid construction | 34 |
| pMO3312 | pET14b plus rex (without start codon, 642 bp); Apr; for Rex expression in BL21(DE3) competent cells | This study |
| pMO3313 | pMO9075 with DVU_0916 (rex) constitutively expressed from Pnpt | 33 |
| pMO9075 | pMO719 containing Pnpt for constitutive expression of complementation constructs; pBG1 stable SRB replicon; Spr | 7 |
| pMO9292 | Spr and pUC ori from pMO719 plus 383-bp upstream and 319-bp downstream DNA regions from Psat (−150–1) flanking the artificial operon of Pnpt-npt-upp from pMO746; for marker exchange mutagenesis; Spr and Kmr | This study |
| pMO9311 | Spr and pUC ori from pMO719 plus 403-bp upstream and 467-bp downstream DNA regions from Psat (−150); wild-type sequence; Spr; for site-directed mutagenesis | This study |
| pMO9313 | Spr and pUC ori from pMO719 plus 403-bp upstream and 467-bp downstream DNA regions from Psat (−150); G−147A Psat; Spr; for site-directed mutagenesis | This study |
| pMO9315 | Spr and pUC ori from pMO719 plus 403-bp upstream and 467-bp downstream DNA regions from Psat (−150); GTA−147–145ACG Psat; Spr; for site-directed mutagenesis | This study |
| pMO9317 | Spr and pUC ori from pMO719 plus 403-bp upstream and 467-bp downstream DNA regions from Psat (−150); CAC−136–134TGT Psat; Spr; for site-directed mutagenesis | This study |
| pMO9319 | Spr and pUC ori from pMO719 plus 403-bp upstream and 467-bp downstream DNA regions from Psat (−150); GTA−147–145ACG Psat CAC−136–134TGT Psat; Spr; for site-directed mutagenesis | This study |
a
Km, kanamycin; Sp, spectinomycin; Ap, ampicillin; 5-FU, 5-fluorouracil; superscript “r” or “s,” resistance or sensitivity, respectively.