TABLE 1.
Bacterial strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant characteristic(s) or sequence(s) (5′-3′)a | Reference or source |
|---|---|---|
| Strains | ||
| Erwinia amylovora | ||
| Ea110 | Wild type, isolated from apple | 49 |
| Ea110− | Ea110, cured of pEA29 | 49 |
| Ea1189 | Wild type, isolated from apple | 17 |
| CFBP1430 | Wild type, isolated from Crataegus | 28 |
| M52 (Ea dspA) | CFBP1430, dspA::uidA-Km, Kmr | 28 |
| Ea110 hrpA | Ea110 ΔhrpA Kmr | 37 |
| ZYC1-3 (Ea hopPtoCEA) | hopPtoC::Km; partial deletion and Kmr insertional mutant of hopPtoCEA of Ea1189; Kmr | This study |
| ZYE3-11 (Ea mltEEA) | mltE::Km; partial deletion and Kmr insertional mutant of mltEEA of Ea1189; Kmr | This study |
| E. coli | ||
| DH10B | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araΔ139 Δ(ara leu)7697 galU galK λ−rpsL (Strr) nupG | Invitrogen |
| S17-1 | recA pro hsdR RP4-2-Tc::Mu-Km::Tn7 | 76 |
| S17-1 λ pir | λ−pir lysogen of S17-1 | 76 |
| Plasmids | ||
| pBluescript II SK(+) | Apr; cloning vector | Stratagene |
| pGem3zf+ | Apr; cloning vector | Promega |
| pCAM140 | Smr Spr Apr; R6K origin; mTn5SSgusA40 | 76 |
| pCAM140-MCSb | Apr; R6K origin; pCAM140 derivative without mini-Tn5; contains the multiple cloning site of pBluescript II SK(+) | 17 |
| pX1918GT | xylE-Gmr fusion cassette-containing plasmid flanked by inverted repeats of the pUC19 MCS | 66 |
| pBSL15 | Km cassette flanked by inverted repeats of the pUC18 MCS | 2 |
| pGCM0 | Gmr cassette with downstream transcriptional terminator and gusA with upstream translational stop codons in pGem3zf+ | This study |
| pZYF2 | 570-bp dspE promoter in opposite orientation relative to uidA in pGCM0 | This study |
| pZYF8 | 570-bp dspE promoter in correct orientation relative to uidA in pGCM0 | This study |
| Primers | ||
| Aj1388 | CCCAAGCTTGGTGCGCCAGGAGAGTTGTTG (HindIII) | |
| Aj1389 | AAAACTGCAGTGATTGATTGACGGACCAGTATTATTATC (PstI) | |
| Aj1390 | CCGGAATTCCGAATTGACATAAGCCTGTTCGG (EcoRI) | |
| Aj1391 | CGGGGTACCTGGACGCGGCCGATCACCTGGCCGTTG (KpnI) | |
| Aj1585 | GATAATAATACTGGTCCGTCAATC | |
| Aj1565 | CGGTTTACAAGCATAAAGCTGGGCAACGGCC | |
| DspE1 | TCCCCCGGGCAGTGAGGGGGGGCAGACTTTTTTTTAACC (SmaI) | |
| DspE2 | TCCCCCGGGTATCTTCGCCGCTGCCACCTTTCACCATTG (SmaI) | |
| PtoC1 | TCCCCGCGGGCGGGCTGTTGGTCTTGCTCT (SacII) | |
| PtoC2 | TGCTCTAGACTCTGGCAAAATTCAACTGA (XbaI) | |
| PtoC3 | CCGGAATTCCATGGCAGGGACCCGCAGTTTG (EcoRI) | |
| PtoC4 | CCGCTCGAGGGCTGATGGCGGGTTAGTCTGTCG (XhoI) | |
| MltE1 | TCCCCGCGGTGAATAGTGCGTGGCGTGATGTGC (SacII) | |
| MltE2 | TGCTCTAGATTAATCATTGCAATCGCCTCGTC (XbaI) | |
| MltE3 | CCGGAATTCTTACCAGCACGTGCAGACAAAACA (EcoRI) | |
| MltE4 | CCGCTCGAGCCGGATGGATCTGGTGAGGGGCGC (XhoI) | |
| AD1 | NTCGASTWTSGWGTT | |
| AD2 | NGTCGASWGANAWGAA | |
| AD3 | WGTGNAGWANCANAGA |
a
Kmr, Apr, Gmr, Spr, and Smr, kanamycin, ampicillin, gentamicin, spectinomycin, and streptomycin resistance, respectively. Underlined nucleotides are restriction sites added, and the restriction enzymes are indicated at the ends of primers. Mixed nucleotides: S, C + G; W, A + T; N, A + T + C + G.
b
MCS, multiple cloning site.