Molecular characterization of natural Erwinia amylovora strains deficient in levan synthesis

Several Erwinia amylovora strains isolated in the Netherlands from apple and pear trees and other hosts for fireblight were found to be conditionally deficient in levan synthesis. The levansucrase genes of five strains were cloned via PCR amplification, and the encoded enzyme was expressed in Escherichia coli. Levan was also produced from the cloned genes in high copy number plasmids, when they were transformed into the corresponding levan-deficient strains. The expression was not driven by the promoter in the cloning vector. An lsc transcript was detected by northern blot analysis with an intermediate signal for strain PD207 and, surprisingly, with a strong signal for PD576. The other four strains did not produce a hybridization signal. The nucleotide sequence in front of the promoter region, when assayed for all five deficient strains, was identical with the sequence previously determined from wild type strain Ea7/74. Also strain E8, which is deficient in levan and in amylovoran synthesis, carries an intact lsc-gene. At 28 degrees C the expression of Ise from the deficient strains was low in Several media with various nutrients and sugars. However, when the strains were grown at 18 degrees C, levan synthesis was partially restored, indicating a temperature-dependent regulation of lsc-expression. (C) 1997 Academic Press Limited.