TABLE 1.
E. coli strains and plasmids used in this study
| Strain or plasmid | Relevant propertiesa | Source and/or reference |
|---|---|---|
| Strains | ||
| MG1655 | F− λ− | 3 |
| ORN225 | MG1655 except fimB′-tetR fimE::IS1 (stably expresses type 1 pili); Hag+ Bag+ | P1 transduction from strain ORN202b |
| ORN226 | ORN225 except fimH′-neoR (stably expresses nonhemagglutinating type 1 pili); Bag+ Hag− | P1 transduction from strain ORN133 (35) |
| ORN227 | MG1655 except Δ(fimBEAICDFGH) ΩneoR; Hag− Bag− | P1 transduction from strain ORN172 (62) |
| ORN201 | thr-1 leuB thi-1 Δ(argF-lac)U169 malAI xyl-7 ara-13 mtl-2 gal-6 rpsL fhuA2 supE44 pilG recA13 Δ(fimBEAICDFGH); Hag− Bag− | 22 |
| Plasmids | ||
| pACYC184 | P15A; Cmr Tcr | 9 |
| pSH2 | pACYC184 fimBEAICDFGH; Cmr (Hag+ Bag+ when resident in strain ORN201) | 21 |
| pORN307 | KpnI-SalI fimH deletion mutant of pSH2; entire fimH gene deleted (Hag− Bag+ when resident in strain ORN201) | 21 |
Hag+, Capable of hemagglutinating guinea pig erythrocytes as described in the text; Bag+, bacterial agglutination in polyclonal rabbit antiserum raised against purified type 1 pili as described in the text; Cmr, chloramphenicol resistant; Tcr, tetracycline resistant.
Strain ORN202 was constructed by first inserting a XhoI DNA fragment containing the tetR gene from Tn10 into the ClaI site in fimB on pSH2 in vitro, creating pORN314. (The ClaI site in fimB had been converted to an XhoI site via linker insertion mutagenesis [45] after partial digestion of pSH2 with ClaI.) Linearization of the plasmid with SalI, followed by transformation ORN117 (ΩfimA′-lacZ fimE::IS1 recBC sbcB) (46), produced a pool of recombinants, some of which had the desired fimE::IS1 fimB′-tetR alleles and were in the locked ON phase (stably transcribing the fimA′-lacZ gene). Such recombinants were identified by phenotypic screening, and the genotype was confirmed by DNA hybridization tests by methods previously described (57). P1 transductions were subsequently used to position the fimE::IS1 fimB′-tetR alleles adjacent to fimA in ORN115 (46), creating the ORN202 donor strain used to construct the stably piliated version of MG1655 (ORN225) used here.