FIG. 4.

Construction of algKΔ mutants of P. aeruginosa. (A) Plasmid pSJ15 contains a 3.8-kb EcoRI (R) fragment of the algD operon in FRD1 cloned into a gene replacement vector (pEX100T), with a nonpolar Gm^r cartridge (hatched arrow) replacing algK. Shown are the predicted genotypes of merodiploids that formed when pSJ15 integrated into the chromosome of mucoid FRD1 to form mucoid (Alg⁺) and nonmucoid (Alg⁻) colonies. When a crossover occurs at site 1, the integrated vector has a polar effect on the transcription of downstream genes (open arrows), and the strain should be Alg⁻. When a crossover occurs at site 2, the promoter of the Gm^r gene should drive transcription of downstream genes in the algD operon so that all genes in the operon are expressed (black arrows), and this strain should be Alg⁺. (B) Diagrams of pSJ44, which expresses algK in trans, and the algD operons in the mutants used in this study along with their colony morphologies are shown. Note that in a strain overproducing alginate (FRD1), a nonpolar algKΔ::Gm^r mutation resulted in a small-colony (i.e., sick) phenotype (strain FRD1100) and that this phenotype could be suppressed (strain FRD1105) with an algJ::Tn501-3 mutation (shown as a pin) which was polar on algA. Open arrows indicate genes that are not expressed. The genes of the algD operon are not drawn to scale.