TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmidPhenotype and/or genotypeaReference or source
P. aeruginosa strains
    FRD1Alg+ CF isolate; contains mutation in mucA 42
    FRD1::pJLS3Prototrophic, Alg+ CF isolate; algD operon controlled by tac promoter; CbrD. Ohman
    FRD2Spontaneous nonmucoid strain derived from FRD1 43
    2-2 algX deletion mutant derivative of FRD1::pJLS3; Gm cassette replaces part of algX; CbrThis study
E. coli strains
    DH10BFmcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL(Strr) endA1 nupG fhuA::IS2Invitrogen
    DH5α supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 51
    JM109 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi Δ(lac-proAB) F′ [traD36 proAB+lacIqlacZΔM15] 51
    HB101 supE44 hsdS20(rB mB) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 51
    HMS174(DE3)pLysSFrecA hsdR (rk12 mk12+) Rifr (DE3) pLysS CmrNovagen
Plasmids
    pALG2-3.3HindIII/XbaIApr; 3.3-kb HindIII-XbaI fragment cloned into pBluescript SK(−); contains part of algG, all of algX, and part of algLThis study
    pBSSK(−)pALG2-2.9ΔXApr; 2.9-kb HindIII-XbaI fragment cloned into pBluescript SK(−); contains part of algG, algX with a 384-bp XcmI fragment deleted from its 3′ end, and part of algLThis study
    pAR4Cbr Gmr; pBSSK(−)pALG2-2.9ΔX with 40-bp EcoRI fragment deleted within algX just upstream of area with 384-bp XcmI fragment replaced with 700-bp Gm nonpolar cassetteThis study
    pAR5Cbr Gmr; 3.6-kb HindIII/XbaI fragment from pAR4 blunted and ligated into pEX100T previously cut with SmaIThis study
    pAR6Tcr; 1.6-kb HindIII/KpnI PCR fragment containing wild-type algX cloned into pRK415This study
    pSJ12Apr Gmr; 700-bp SmaI fragment containing a nonpolar Gm cassette cloned into pBluescript II KS(−) 28
    pEX100T bla (Cbr Apr) sacB oriT 55
    pRK415Broad-host-range expression vector; contains lac promoter; Tcr; mob+ 30
    pRK2013Kmr ColE1-Tra(RK2)+ 13
    pNLS20pRK415 vector carrying 2.5-kb fragment containing algX and oriented for its expressionN. Schiller
    pSM7pET21a vector with algX for expression of AlgX-His; inserted via NdeI/XhoI sitesThis study
    pET-21aExpression vector; Apr; T7lac promoter; product contains His tag at C-terminal endNovagen
    pBluescript SK(−)Apr; ColE1 originStratagene
  • a Abbreviations: Alg+, alginate producing; Cbr, carbenicillin resistance; Cmr, chloramphenicol resistance; Apr, ampicillin resistance; Tcr, tetracycline resistance; mob+, mobilizable plasmid; Kmr, kanamycin resistance.