Construction and Characterization of an Erwinia chrysanthemi Mutant with Directed Deletions in All of the Pectate Lyase Structural Genes. Jeffrey L. Ried. Department of Botany and Agricultural Biotechnology Center of the Maryland Biotechnology Institute, University of Maryland, College Park, MD 20742, U.S.A.. Alan Collmer. Department of Botany and Agricultural Biotechnology Center of the Maryland Biotechnology Institute, University of Maryland, College Park, MD 20742, U.S.A.. MPMI 1:32-38. Accepted 21 August 1987. Copyright 1987 The American Phytopathological Society.

Erwinia chrysanthemi EC16 produces four isozymes of pectate lyase (PL), an extracellular enzyme that macerates parenchymatous plant tissues. The pelB and pelC genes, which encode isozymes PLb and PLc, were deleted from the chromosome by marker exchange-eviction mutagenesis: an nptI-sacB-sacR cartridge, encoding kanamycin resistance and sucrose sensitivity, was inserted in a cloned E. chrysanthemi DNA fragment in place of the pelB and pelC genes; the marked deletion was then introduced into the chromosome by exchange recombination (selecting for kanamycin resistance) and subsequently evicted by a second recombinational exchange with an unmarked deletion derivative (selecting for sucrose tolerance). The resulting mutant, UM1003, was deficient in PLb and PLc and kanamycin-sensitive. The pelA and pelE genes, carried on adjacent EcoRI fragments in another E. chrysanthemi DNA clone, were replaced with an nptI cartridge and then exchanged into the UM1003 chromosome by selecting for kanamycin resistance. Mutant UM1005 was deficient in PLa, PLb, PLc, and PLe and produced less than 0.1% of the extracellular PL activity of the wild type. However, the specific growth rate of UM1005 was unchanged relative to the wild type in a minimal medium with polygalacturonic acid as the sole carbon source. Furthermore, although virulence was reduced 79-98% (depending on the assay) in potato tuber maceration tests, mutant UM1005 was still able to cause significant maceration in potato, carrot, and pepper tissues. These observations indicate that PL is not necessary for the utilization of pectate or the maceration of plant tissues by E. chrysanthemi.