Use of Mu-lac Insertions to Study the Secretion of Pectate Lyases by Erwinia chrysanthemi

Members of the species Erwinia chrysanthemi are phytopathogenic enterobacteria causing soft-rot disease due, in part, to the secretion of pectolytic enzymes. We studied the secretion of pectate lyases by using Mu-lac insertion mutagenesis in E. chrysanthemi strain 3937. Analysis of β-galactosidase expression of the out—lac fusions, in different growth conditions, showed that the expression of the out gene is constitutive. Compartmentation of pectate lyases during growth suggests that these enzymes are first exported to the periplasm with formation of an intracellular pool of active pectate lyases and then released into the extracellular medium. In the out mutants, pectate lyases are retained in the periplasm. Analysis by electrofocusing showed that the pectate lyases are not modified during the transfer via the outer membrane. A 65 kDa protein is absent from the periplasm of the out mutants; this protein could have a role in the secretory system of E. chrysanthemi.