TABLE 1.
Strains and plasmids used
| Strain or plasmid | Descriptiona | Reference or source |
|---|---|---|
| E. corrodens strains | ||
| VA1 | Clinical isolate | Laboratory collection |
| VA1-S1 | S-phase variant of VA1 | Laboratory collection |
| VA1-L2 | L-phase variant of VA1 | Laboratory collection |
| T18 | Derivative of VA1-S1; pilA1::Ω | This study |
| T6 | Derivative of VA1-S1; pilA2::Ω | This study |
| T11 | Derivative of VA1-S1; pilB::Ω | This study |
| T40 | Derivative of VA1-S1; hagA::Ω | This study |
| T99 | Derivative of VA1-S1; ΔpilA1 pilB::Ω | This study |
| E. coli DH5α | F− φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK− mK+) supE44 λ− thi-1 gyrA96 relA1 | Bethesda Research Labs |
| Plasmids | ||
| pHP45Ω | Source of aad gene (Ω cassette); Spr | 22 |
| pSKS101 | Source of aph gene; Kmr | 27 |
| pUC1819EcoRI | Modified pUC18 cloning vector; Apr | J. Golden |
| pUMC315 | pUC1819 with 2.0-kbp EcoRI fragment from pHP45Ω containing the Ω cassette | This study |
| pEC114 | pGEM3zf(+) containing the 3.9-kbp EcoRI fragment from VA1-S1 carrying the pilA locus (pilA1A2B hagA) | 31 |
| pEC207 | pEC114 deleted for 299-bp HindIII fragment (nucleotides 3654–3953) | This study |
| pEC211 | pEC114 with BamHI-digested Ω cassette inserted into BclI site at nucleotide 1327 in pilA2; pilA2::Ω | This study |
| pEC213 | pEC114 with BamHI site replacing BglII site at nucleotide 869 in pilA1 | This study |
| pEC216 | pEC213 with BamHI-digested Ω cassette inserted into BglII site at nucleotide 2008 in pilB; pilB::Ω | This study |
| pEC218 | pEC114 with BamHI site replacing BglII site at nucleotide 2008 in pilB | This study |
| pEC219 | pEC218 with BamHI-digested Ω cassette inserted into BglII site at nucleotide 869 in pilA1; pilA1::Ω | This study |
| pEC230 | pEC207 with 1575-bp SmaI-BglII fragment (nucleotides 432-2008) replaced with 1,035-bp PCR product from pEC114; ΔpilA1; pilA2 controlled by pilA1p | This study |
| pEC232 | pEC230 with BamHI-digested Ω cassette inserted into BglII site at nucleotide 2008 in pilB; ΔpilA1; pilA2 controlled by pilA1p; pilB::Ω | This study |
| pEC233 | pEC230 with BamHI-digested aph gene inserted into BglII site at nucleotide 2008 in pilB; ΔpilA1; pilA2 controlled by pilA1p; pilB::aph | This study |
| pEC235 | pEC230 with SalI-digested Ω cassette inserted into SalI site at nucleotide 3274 in hagA; ΔpilA1; pilA2B controlled by pilA1p; hagA::Ω | This study |
| pEC237 | pEC230 with SalI-digested aph gene inserted into SalI site at nucleotide 3274 in hagA; ΔpilA1; pilA2B controlled by pilA1p; hagA::aph | This study |
| pEC306 | pEC114 with SalI-digested Ω cassette inserted into SalI site at nucleotide 3274 in hagA; hagA::Ω | This study |
a
Nucleotide numbers correspond to pilA locus sequence deposited in GenBank (accession no. AF079304).