Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa

Abstract

Mucoid strains of P. aeruginosa isolated from the sputum of cystic fibrosis patients produce copious quantities of an exopolysaccharide known as alginic acid. Since clinical isolates of the mucoid variants are unstable with respect to alginate synthesis and revert spontaneously to the more typical nonmucoid phenotype, it was difficult to isolate individual structural gene mutants defective in alginate synthesis. The cloning of the genes controlling alginate-producing strain, 8830. The stable mucoid strain was mutagenized with ethyl methanesulfonate to obtain various mutants defective in alginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P. aeruginosa gene library was then constructed, using a cosmid cloning vector. DNA isolated from the stable mucoid strain 8830 was partially digested with the restriction endonuclease hindIII and ligated to the HindIII site of the broad host range cosmid vector, pCP13. After packaging in phage .lambda. particles, the recombinant DNA was introduced via transfection into Escherichia coli AC80. The clone bank was mated (en masse) from E. coli into various P. aeruginosa 8830 nonmucoid mutants with the help of pRK2013, which provided donor functions in trans, and tetracycline-resistant exconjugants were screened for the ability to form mucoid colonies. Three recombinant plasmids, pAD1, pAD2 and pAD3, containing DNA inserts at 20, 9.5, and 6.2 kilobases [kb] respectively, were isolated based on their ability to restore al alginate synthesis in various strain 8830 nonmucoid (Alg-) mutants. Mutants were assigned to at least 4 complementation groups, based on complementation by pAD1, pAD2, or pAD3 or by none of them. Introduction of pAD1 into the spontaneous nonmucoid strain 8822, and into other nonmucoid laboratory strains of P. aeruginosa such as PAO and SB1, was found to slowly induce alginate synthesis. This alginate-inducing ability was found to reside on a 7.5-kb EcoRI fragment that complemented the alg-22 mutation of strain 8852. The pAD1 chromosomal insert which complements the alg-22 mutation was subsequently mapped at .apprx. 19 min of the P. aeruginosa PAO chromosome.