Cloning ofGenesControlling Alginate Biosynthesis froma Mucoid Cystic Fibrosis Isolate ofPseudomonas aeruginosa

knownasalginic acid. Since clinical isolates ofthemucoidvariants are unstable withrespect toalginate synthesis andrevert spontaneously tothemore typical nonmucoid phenotype, ithasbeendifficult toisolate individual structural genemutantsdefective inalginate synthesis. The cloning ofthegenescontrolling alginate synthesis hasbeenfacilitated bytheisolation ofa stable alginateproducing strain, 8830.Thestable mucoidstrain was mutagenized withethyl methanesulfonate toobtain various mutantsdefective inalginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P.aeruginosa genelibrary was thenconstructed, using acosmid cloning vector. DNA isolated fromthestable Mpucoid strain 8830 'waspartially digested withtherestriction endonuclease HindIUandligated totheHindll site ofthebroad -host rangecosmid yector, pCP13.After packaging inlambdaparticles, therecombinant DNA wasintroduced viatransfection into Escherichia coliAC80.Theclone bankwas mated(enmasse) fromE.coli into various P.aeruginosa 8830nonmucoid mutants withthehelpofpRK2013, whichprovided donorfunctions intrans, andtetracycline-resistant exconjugants were screened fortheability toformmucoidcolonies. Three recombinant plasmids, pAD1,pAD2,andpAD3,containing DNA inserts of20,9.5,and6.2kilobases, respectively, were isolated based on their ability torestore alginate synthesis invarious strain 8830nonmucoid (Alg-) mutants. Mutants havebeenassigned toatleast fourcomplementation groups,based on complementationbypAD1,pAD2,or pAD3orbynoneofthem.Introduction ofpAD1intothespontaneous nonmucoid strain 8822, aswell asintoother nonmucoid laboratory strains ofP.aeruginosa suchasPAO andSB1,was foundtoslowly induce alginate synthesis. Thisalginate-inducing ability was foundtoreside on a 7.5-kilobase EcoRIfragment thatcomplemented thealg-22 mutation ofstrain 8852. ThepAD1chromosomal insert which complements thealg-22 mutation was subsequently mappedatca. 19minoftheP.aeruginosa PAO chromuosome.