TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmidRelevant characteristic(s)a
Strains
    E. coli
        DH5αendA1 hsdR17(rK mK+) glnV44 thi-1 recA1 gyrA relA1 Δ(lacIZYA-argF)U16980dlacΔ(lacZ)M15]
        S17-1thi pro hsdR recA chr::RP4-2 (45)
    P. aeruginosa
        PAKWild-type P. aeruginosa strain Kb
        PAO1Wild-type P. aeruginosa strain ATCC 15692
        PAO1293Cmr derivative of P. aeruginosa PAO1
        PAKpilA::TcPreviously referred to as AWK (53)
        118C8PAO1293 mTn5-Tc vfr mutant identified during transposon library screen
        PAKvfr::mTn 5-TcPAK with same mTn5-Tc insertion as 118C8
        PAO1vfr::TcPAO1 with Tc cartridge inserted as blunt fragment into unique BclI site of vfr
        R306PAK Tn5-B21 pilV mutant (21)
Plasmids
    pSM-TETSource of Tcr cassette (31)
    pRIC380P. aeruginosa suicide vector (4)
    pOK12Kmr cloning vector (50)
    pUCPSKP. aeruginosa-E. coli shuttle vector (52)
    pBluescript II SKE. coli cloning vector with Ap selection (Stratagene)
    pMO011925pLA2917 cosmid from Holloway library found to contain vfr (56)
    pUCPvfrA1.3-kb XhoI fragment containing vfr cloned from pMO011925 into pUCPSK
    pUCPvfrF1.3-kb XhoI fragment containing vfr cloned from pMO011925 into pUCPSK
    pSB77.12Marker rescue clone from PstI digest of 118C8 (carries vfr::mTn 5-Tc insert)
    pSB172.116.2-kb PstI insert (including 2.2-kb mTn 5-Tc) from pSB77.12 cloned into pOK12
    pSB172.26.2-kb SpeI insert from pSB172.11 cloned into pRIC380
    pSB201.13ApUCPvfrA with Tcr cassette from pSM-TET inserted within blunted BclI site
    pSB206.1A4.3-kb NotI-KpnI insert (including 3-kb Tcr cassette) from pSB201.13A cloned into pOK12
    pSB216.1A4.3-kb SpeI insert from pSB206.1A cloned into pRIC380
    pSB299.15Avfr amplified from wild-type PAO1 and cloned into pUCPSK against lac
    pSB299.17Avfr amplified from PAK lasI-RT and cloned into pUCPSK against lac
  • a Antibiotic resistance abbreviations: Tc, tetracycline; Cm, chloramphenicol; Km, kanamycin; Ap, ampicillin.

  • b Source: David Bradley, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.