Genetics of the Damage Inducible Phenotypes of Erwinia Carotovora Subsp. Carotovora: Characterization of Tn5 Insertional Mutants Defective in Pectin Lyase and Carotovoricin

Abstract

Carotovoricin (CTV) and pectin lyase (PNL) are coinduced in the soft-rot organism Erwinia carotovora subsp. carotovora (Ecc) by DNA damaging agents such as bleomycin, mitomycin C, nalidixic acid or ultraviolet irradiation. We have recently shown that these recA-dependent phenotypes are also expressed in potato tuber tissue. To determine the organization of ctv and pnl, a genetic analysis of Ecc strain 71 was undertaken. Development of plate assays for PNL and CTV allowed Tn5 insertion mutants defective in CTV, PNL or both to be isolated. Mutants of these three phenotypic classes contained a single copy of Tn5 located in different regions of the Ecc genome. Phenotypes of the mutants, including the wild-type levels of pectolytic enzymes, were confirmed in agar and broth cultures and in potato tuber tissue. Since Ctv^- Pnl⁺ and Ctv⁺ Pnl^- mutants were obtained, and Tn5 produces strong polar mutations within operons, ctv and pnl most likely are components of independent transcriptional units inducible by a common signal. The Ctv^- Pnl^- phenotype resembles that of a RecA strain of Ecc except that (a) this class of Tn5 mutants undergoes typical wild-type filamentation after UV irradiation or addition of mitomycin C, contrasting the RecA mutant which does not filament, and (b) the mutation could not be complemented by the cloned Ecc recA+ gene. Cloning of the Tn5 carrying fragments and their corresponding wild type alleles is underway which will facilitate further studies involving the organization and regulation of ctv and pnl.