TABLE 1.
Bacterial strains and plasmids
| Strain or plasmid | Genotype and/or phenotypea | Source or reference |
|---|---|---|
| E. coli | ||
| HB101 | proA2 leuB6 thi-1 lacY1 hsdR hsdM recA13 supE44 rpsL20 | This laboratory |
| JM109(DE3) | F′ traD36 lacIq Δ(lacZ)M15 proA+ B+/e14− (McrA−) Δ(lac-proAB) thi gyrA96 endA1 hsdR17 (rK− mK+) relA1 supE44 recA1 with DE3, a λ prophage carrying the T7 RNA polymerase gene under control of PlacUV5 | |
| P. aeruginosa | ||
| FRD1 | Cystic fibrosis isolate, Alg+ | 28 |
| FRD1003 | algJ::Tn501-3 Hgr Alg− | 13 |
| FRD1100 | algKΔ::Gmr Alg− | This study |
| FRD1105 | algKΔ::GmralgJ::Tn501-3 Hgr Alg− | This study |
| PAO1 | Prototrophic wild-type strain (nonmucoid) | 19 |
| Plasmids | ||
| pALG2 | pBR322 cos oriT Tn5 Apr Kmr with 35 kb of P. aeruginosa DNA containing the alginate biosynthetic operon and argF | 5 |
| M13mp18 | Derivative of M13 | New England Biolabs |
| pBluescript II KS(−) | ColE1 oriV lacZa Apr | Stratagene |
| pCC75 | pMMB22 (IncQ) with Ptac control of algA on a 2.0-kb fragment | 6 |
| pEX100T | bla (Cb/Apr) sacB oriT | 40 |
| pPHO7 | P15A oriV (Tcr) phoA (promoterless) | 18 |
| pMF54 | Ptrc ColE1 replicon Apr with oriV-SF oriT(RK2) lacIq | 11 |
| pRK2013 | ColE1-Tra(RK2)+ Kmr | 10 |
| pSJ5 | M13mp18 with 1.2-kb KpnI fragment from pALG2 | This study |
| pMF100 | pBluescript II KS(−) with PT7 control of alg44 algK on a 3.8-kb EcoRI fragment from pALG2 | This study |
| pSJ10 | pEX100T with alg44 algK on a 3.8-kb EcoRI fragment from pMF100 | This study |
| pSJ12 | pBluescript II KS(−) with 0.7-kb SmaI fragment containing a nonpolar Gmr cartridge | This study |
| pSJ15 | pSJ10 with algKΔ::Gmr (i.e., 1.2-kb KpnI fragment replaced by SmaI fragment from pSJ12) | This study |
| pSJ42 | pMF54 with the N-terminal 360-bp NcoI-XbaI fragment of algK | This study |
| pSJ43 | pMF54 with the N-terminal 1,020-bp NcoI-XbaI fragment of algK | This study |
| pSJ44 | pMF54 with alg44 algK on a 3.8-kb EcoRI fragment | This study |
| pSJ46 | pSJ42 with an algK (1-120)-phoA protein fusion by insertion of a 2.3-kb XbaI-XhoI fragment from pPHO7 | This study |
| pSJ47 | pSJ43 with an algK (1-340)-phoA protein fusion by insertion of a 2.3-kb XbaI-XhoI fragment from pPHO7 | This study |
| pSJ137 | pBluescript II KS(−) with PT7 control of alg44 algKΔ::Gmr | This study |
| pSJ151 | pMF54 with Ptac control of alg44 algKΔ::Gmr | This study |
a
Apr, ampicillin resistance; Cbr, carbenicillin resistance; Tcr, tetracycline resistance; Kmr, kanamycin resistance; Hgr, mercury resistance; Gmr, gentamicin resistance; Alg+, alginate overproduction.