Cloning and Characterization of iaaM and iaaH from Erwinia herbicola pathovar gypsophilae

Erwinia herbicola pv. gypsophilae induces galls on its host, Gypsophila paniculata. A 16-kb DNA fragment derived from a 78-Md native plasmid with homology to the iaa operon of Pseudomonas syringae pv. savaslanoi was isolated from an EMBL3 library of E. h. gypsophilae, strain PD713, DNA. A 7.5-kb £coRI fragment was subcloned into pUCI 18 to generate pEGlOI. Escherichia coli DH5o cells transformed with pEGIOI produced indole-3-acetic acid (IAA) when cultured in medium supplemented with i.-tryptophan (TRP). Pcrmcabilized, transformed cells direct the synthesis of IAA from indolc-3-acetamide (IAM). The IAA biosynlhetic capability was localized to a 4.0-kb ///ndIII-£coRI fragment through subcloning and insertional inactivation. The IAA biosynlhetic genes of E. h. gypsophilae were designated iaaM and iaaH because of their structural and functional similarity to the iaaM and iaaH of P. s. savaslanoi, which encode tryptophan-2-monooxygenase and indoleacetamide hydrolase, respectively. Insertional mutations were generated in £. h. gypsophilae iaaM and iaaH. Marker-exchange mutants of E. h. gypsophilae, generated using insertionally inactivated constructs, produced the same amount of IAA in culture as the wild type. The marker-exchange mutants, which exhibited either reduction or elimination of iaaH activity, induced smaller galls than did unmodified E. h. gypsophilae.