MOLECULAR-CLONING OF AN AEPA GENE THAT ACTIVATES PRODUCTION OF EXTRACELLULAR PECTOLYTIC, CELLULOLYTIC, AND PROTEOLYTIC-ENZYMES IN ERWINIA-CAROTOVORA SUBSP CAROTOVORA

Strain 71 of Erwinia carotovora subsp. carotovora produces extracellular enzymes such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The levels of extracellular Pel, Cel, and Prt were higher in a medium containing crude celery extract than in a medium containing pectate. Using transposons (Tn5, TnphoA, and Tn10-lacZ), we isolated pleiotropic mutants that were deficient in extracellular levels of these enzymes and attenuated in their ability to macerate plant tissues. The mutants, however, were similar to the parent in their ability to utilize various sugars and to produce periplasmic enzymes. In an E. c. subsp. carotovora 71 gene library, we detected a cosmid, pAKC264, that restored extracellular enzyme production and tissue maceration in all the mutants. The cosmid appears not to carry pel, peh, cel, or prt genes. In E. c. subsp. carotovora 71, pAKC264 stimulated the production of Pel, Peh, Cel, and Prt, but it did not affect the levels of the periplasmic enzymes, cyclic phosphodiesterase, or beta-lactamase. pAKC602, a subclone of pAKC264, stimulated enzyme production in E. c. subsp. carotovora 71 and did not complement mutations in cya, the gene specifying adenylate cyclase, or crp, the gene specifying cyclic AMP receptor protein, in Escherichia coli. The E. c. subsp. carotovora 71 gene that activates extracellular protein production was designated as aepA. Sixteen mini-Mu-lacZ (MudI1734) insertions inactivating aepA spanned a DNA region of about 0.8 kilobases and allowed determination of the direction of aepA transcription by screening for beta-galactosidase production. By further subcloning and localizing the sites of mini-Mu-lacZ insertions not inactivating aepA, the gene was localized within a 1.1-kilobase DNA segment.